Treatment of Clostridium difficile induced disease

ABSTRACT

The present provides neutralizing antitoxin directed against C. difficile toxins. These antitoxins are produced in arian species using soluble recombinant C. difficile toxin proteins. The avian antitoxins are designed so as to be orally administrable in therapeutic amounts and may be in any form (i.e., as a solid or in aqueous solution). Solid forms of the antitoxin may comprise an enteric coating. These antitoxins are useful in the treatment of humans and other animals intoxicated with at least one bacterial toxin. The invention further provides vaccines capable of protecting a vaccinated recipient from the morbidity and mortality associated with C. difficile infection. These vaccines are useful for administration to humans and other animals at risk of exposure to C. difficile toxins.

This application is a Continuation-in-Part of application Ser. No. 08/422,711 filed Apr. 14, 1995, which is a Continuation-in-Part of application Ser. No. 08/405,496 filed Mar. 16, 1995, which is a Continuation-in-Part of application Ser. No. 08/329,154 filed, Oct. 24, 1994, which is a Continuation-in-Part of application Ser. No. 08/161,907, filed on Dec. 2, 1993, now U.S. Pat. No. 5,601,823, which is a Continuation-in-Part of application Ser. No. 07/985,321, filed Dec. 4, 1992, which is a Continuation-in-Part of application Ser. No. 429,791, filed Oct. 31, 1989, now issued as U.S. Pat. No. 5,196,193 on Mar. 23, 1993.

FIELD OF THE INVENTION

The present invention relates to clostridial antitoxin and vaccine therapy for humans and other animals. Antitoxins which neutralize the pathologic effects of clostridial toxins are provided. Vaccines which prevent the morbidity and mortality associated with clostridial diseases are provided.

BACKGROUND OF THE INVENTION

The genus Clostridium is comprised of gram-positive, anaerobic, spore-forming bacilli. The natural habitat of these organisms is the environment and the intestinal tracts of humans and other animals. Indeed, clostridia are ubiquitous; they are commonly found in soil, dust, sewage, marine sediments, decaying vegetation, and mud. See e.g., P. H. A. Sneath et al., "Clostridium," Bergey's Manual® of Systematic Bacteriology, Vol. 2, pp. 1141-1200, Williams & Wilkins (1986).! Despite the identification of approximately 100 species of Clostridium, only a small number have been recognized as etiologic agents of medical and veterinary importance. Nonetheless, these species are associated with very serious diseases, including botulism, tetanus, anaerobic cellulitis, gas gangrene, bacteremia, pseudomembranous colitis, and clostridial gastroenteritis. Table 1 lists some of the species of medical and veterinary importance and the diseases with which they are associated. As virtually all of these species have been isolated from fecal samples of apparently healthy persons, some of these isolates may be transient, rather than permanent residents of the colonic flora.

                  TABLE 1                                                          ______________________________________                                         Clostridium Species of Medical and Veterinary Importance*                      Species       Disease                                                          ______________________________________                                         C. aminovalericum                                                                            Bacteriuria (pregnant women)                                     C. argentinense                                                                              Infected wounds; Bacteremia; Botulism;                                         Infections of amniotic fluid                                     C. baratii    Infected war wounds; Peritonitis; Infectious                                   processes of the eye, ear and prostate                           C. beijerinckikii                                                                            Infected wounds                                                  C. bifermentans                                                                              Infected wounds; Abscesses; Gas Gangrene;                                      Bacteremia                                                       C. botulinum  Food poisoning; Botulism (wound, food,                                         infant)                                                          C. butyricum  Urinary tract, lower respiratory tract, pleural                                cavity, and abdominal infections; Infected                                     wounds; Abscesses; Bacteremia                                    C. cadaveris  Abscesses; Infected wounds                                       C. carnis     Soft tissue infections; Bacteremia                               C. chauvoei   Blackleg                                                         C. clostridioforme                                                                           Abdominal, cervical, scrotal, pleural, and                                     other infections; Septicemia; Peritonitis;                                     Appendicitis                                                     C. cochlearium                                                                               Isolated from human disease processes, but                                     role in disease unknown.                                         C. difficile  Antimicrobial-associated diarrhea; Pseudo-                                     membranous enterocolitis; Bacteremia;                                          Pyogenic infections                                              C. fallax     Soft tissue infections                                           C. ghnoii     Soft tissue infections                                           C. glycolicum Wound infections; Abscesses; Peritonitis                         C. hastiforme Infected war wounds; Bacteremia; Abscesses                       C. histolyticum                                                                              Infected war wounds; Gas gangrene;                                             Gingival plaque isolate                                          C. indolis    Gastrointestinal tract infections                                C. innocuum   Gastrointestinal tract infections; Empyema                       C. irregulare Penile lesions                                                   C. leptum     Isolated from human disease processes, but                                     role in disease unknown.                                         C. limosum    Bacteremia; Peritonitis; Pulmonary infections                    C. malenominatum                                                                             Various infectious processes                                     C. novyi      Infected wounds; Gas gangrene; Blackleg,                                       Big head (ovine); Redwater disease (bovine)                      C. oroticum   Urinary tract infections; Rectal abscesses                       C. paraputrificum                                                                            Bacteremia; Peritonitis; Infected wounds;                                      Appendicitis                                                     C. perfringens                                                                               Gas gangrene; Anaerobic cellulitis; Intra-                                     abdominal abscesses; Soft tissue infections;                                   Food poisoning; Necrotizing pneumonia;                                         Empyema; Meningitis; Bacteremia; Uterine                                       Infections; Enteritis necrotans; Lamb                                          dysentery; Struck; Ovine Enterotoxemia;                          C. putrefaciens                                                                              Bacteriuria (Pregnant women with                                               bacteremia)                                                      C. putrificum Abscesses; Infected wounds; Bacteremia                           C. ramosum    Infections of the abdominal cavity, genital                                    tract, lung, and biliary tract; Bacteremia                       C. sartagoforme                                                                              Isolated from human disease processes, but                                     role in disease unknown.                                         C. septicum   Gas gangrene; Bacteremia; Suppurative                                          infections; Necrotizing enterocolitis; Braxy                     C. sordellii  Gas gangrene; Wound infections; Penile                                         lesions; Bacteremia; Abscesses; Abdominal                                      and vaginal infections                                           C. sphenoides Appendicitis; Bacteremia; Bone and soft                                        tissue infections; Intraperitoneal infections;                                 Infected war wounds; Visceral gas gangrene;                                    Renal abscesses                                                  C. sporogenes Gas gangrene; Bacteremia; Endocarditis;                                        central nervous system and pleuropulmonary                                     infections; Penile lesions; Infected war                                       wounds; Other pyogenic infections                                C. subterminale                                                                              Bacteremia; Empyema; Biliary tract, soft                                       tissue and bone infections                                       C. symbiosum  Liver abscesses; Bacteremia; Infections                                        resulting due to bowel flora                                     C. tertium    Gas gangrene; Appendicitis; Brain abscesses;                                   Intestinal tract and soft tissue infections;                                   Infected war wounds; Periodontitis;                                            Bacteremia                                                       C. tetani     Tetanus; Infected gums and teeth; Corneal                                      ulcerations; Mastoid and middle ear                                            infections; Intraperitoneal infections; Tetanus                                neonatorum; Postpartum uterine infections;                                     Soft tissue infections, especially related to                                  trauma (including abrasions and lacerations);                                  Infections related to use of contaminated                                      needles                                                          C. thermosaccharolyticum                                                                     Isolated from human disease processes, but                                     role in disease unknown.                                         ______________________________________                                         *Compiled from P. G. Engelkirk et al. "Classification", Principles and         Practice of Clinical Anaerobic Bacteriology, pp. 22-23, Star Publishing        Co., Belmont, CA (1992); J. Stephen and R. A. Petrowski, "Toxins Which         Traverse Membranes and Deregulate Cells," in Bacterial Toxins, 2d ed.,         pp. 66-67, American Society for Microbiology (1986); R. Berkow and             A. J. Fletcher (eds.), "Bacterial Diseases," Merck Manual of Diagnosis         and Therapy, l6th ed., pp. 116-126, Merck Research Laboratories,               Rahway, N.J. (1992); and O. H. Sigmund and C. M. Fraser (eds.),                "Clostridial Infections," Merck Veterinary Manual, 5th ed., pp. 396-409,       Merck & Co., Rahway, N.J. (1979).                                         

In most cases, the pathogenicity of these organisms is related to the release of powerful exotoxins or highly destructive enzymes. Indeed, several species of the genus Clostridium produce toxins and other enzymes of great medical and veterinary significance. C. L. Hatheway, Clin. Microbiol. Rev. 3:66-98 (1990).!

Perhaps because of their significance for human and veterinary medicine, much research has been conducted on these toxins, in particular those of C. botulinum and C. difficile.

C. botulinum

Several strains of Clostridium botulinum produce toxins of significance to human and animal health. C. L. Hatheway, Clin. Microbiol. Rev. 3:66-98 (1990).! The effects of these toxins range from diarrheal diseases that can cause destruction of the colon, to paralytic effects that can cause death. Particularly at risk for developing clostridial diseases are neonates mad humans and animals in poor health (e.g., those suffering from diseases associated with old age or immunodeficiency diseases).

Clostridium botulinum produces the most poisonous biological toxin known. The lethal human dose is a mere 10⁻⁹ mg/kg bodyweight for toxin in the bloodstream. Botulinal toxin blocks nerve transmission to the muscles, resulting in flaccid paralysis. When the toxin reaches airway and respiratory muscles, it results in respiratory failure that can cause death. S. Arnon, J. Infect. Dis. 154:201-206 (1986).!

C. botulinum spores are carried by dust and are found on vegetables taken from the soil, on fresh fruits, and on agricultural products such as honey. Under conditions favorable to the organism, the spores germinate to vegetative cells which produces toxin. S. Arnon, Ann. Rev. Med. 31:541 (1980).!

Botulism disease may be grouped into four types, based on the method of introduction of toxin into the bloodstream. Food-borne botulism results from ingesting improperly preserved and inadequately heated food that contains botulinal toxin. There were 355 cases of food-borne botulism in the United States between 1976 and 1984. K. L. MacDonald et al., Am. J. Epidemiol. 124:794 (1986).! The death rate due to botulinal toxin is 12% and can be higher in particular risk groups. C. O. Tacket et al., Am. J. Med. 76:794 (1984).! Wound-induced botulism results from C. botulinum penetrating traumatized tissue and producing toxin that is absorbed into the bloodstream. Since 1950, thirty cases of wound botulism have been reported. M. N. Swartz, "Anaerobic Spore-Forming Bacilli: The Clostridia," pp. 633-646, in B. D. Davis et al., (eds.), Microbiology, 4th edition, J. B. Lippincott Co. (1990).! Inhalation botulism results when the toxin is inhaled. Inhalation botulism has been reported as the result of accidental exposure in the laboratory E. Holzer, Med. Klin. 41:1735 (1962)! and could arise if the toxin is used as an agent of biological warfare D. R. Franz et al., in Botulinum and Tetanus Neurotoxins, B. R. DasGupta, ed., Plenum Press, New York (1993), pp. 473-476!. Infectious infant botulism results from C. botulinum colonization of the infant intestine with production of toxin and its absorption into the bloodstream. It is likely that the bacterium gains entry when spores are ingested and subsequently germinate. S. Arnon, J. Infect. Dis. 154:201 (1986).! There have been 500 cases reported since it was first recognized in 1976. M. N. Swartz, supra.!

Infant botulism strikes infants who are three weeks to eleven months old (greater than 90% of the cases are infants less than six months). S. Arnon, J. Infect. Dis. 154:201 (1986).! It is believed that infants are susceptible, due, in large part, to the absence of the full adult complement of intestinal microflora. The benign microflora present in the adult intestine provide an acidic environment that is not favorable to colonization by C. botulinum. Infants begin life with a sterile intestine which is gradually colonized by microflora. Because of the limited microflora present in early infancy, the intestinal environment is not as acidic, allowing for C. botulinum spore germination, growth, and toxin production. In this regard, some adults who have undergone antibiotic therapy which alters intestinal microflora become more susceptible to botulism.

An additional factor accounting for infant susceptibility to infectious botulism is the immaturity of the infant immune system. The mature immune system is sensitized to bacterial antigens and produces protective antibodies. Secretory IgA produced in the adult intestine has the ability to agglutinate vegetative cells of C. botulinum. S. Arnon, J. Infect. Dis. 154:201 (1986).! Secretory IgA may also act by preventing intestinal bacteria and their products from crossing the cells of the intestine. S. Arnon, Epidemiol. Rev. 3:45 (1981).! The infant immune system is not primed to do this.

Clinical symptoms of infant botulism range from mild paralysis, to moderate and severe paralysis requiring hospitalization, to fulminant paralysis, leading to sudden death. S. Arnon, Epidemiol. Rev. 3:45 (1981).!

The chief therapy for severe infant botulism is ventilatory assistance using a mechanical respirator and concurrent elimination of toxin and bacteria using cathartics, enemas, and gastric lavage. There were 68 hospitalizations in California for infant botulism in a single year with a total cost of over $4 million for treatment. T. L. Frankovich and S. Arnon, West. J. Med. 154:103 (1991).!

Different strains of Clostridium botulinum each produce antigenically distinct toxin designated by the letters A-G. Serotype A toxin has been implicated in 26% of the cases of food botulism; types B, E and F have also been implicated in a smaller percentage of the food botulism cases H. Sugiyama, Microbiol. Rev. 44:419 (1980)!. Wound botulism has been reportedly caused by only types A or B toxins H. Sugiyama, supra!. Nearly all cases of infant botulism have been caused by bacteria producing either type A or type B toxin. (Exceptionally, one New Mexico case was caused by Clostridium botulinum producing type F toxin and another by Clostridium botulinum producing a type B-type F hybrid.) S. Arnon, Epidemiol. Rev. 3:45 (1981).! Type C toxin affects waterfowl, cattle, horses and mink. Type D toxin affects cattle, and type E toxin affects both humans and birds.

A trivalent antitoxin derived from horse plasma is commercially available from Connaught Industries Ltd. as a therapy for toxin types A, B, and E. However, the antitoxin has several disadvantages. First, extremely large dosages must be injected intravenously and/or intramuscularly. Second, the antitoxin has serious side effects such as acute anaphylaxis which can lead to death, and serum sickness. Finally, the efficacy of the antitoxin is uncertain and the treatment is costly. C. O. Tacket et al., Am. J. Med. 76:794 (1984).!

A heptavalent equine botulinal antitoxin which uses only the F(ab')2 portion of the antibody molecule has been tested by the United States Military. M. Balady, USAMRDC Newsletter, p. 6 (1991).! This was raised against impure toxoids in those large animals and is not a high titer preparation.

A pentavalent human antitoxin has been collected from immunized human subjects for use as a treatment for infant botulism. The supply of this antitoxin is limited and cannot be expected to meet the needs of all individuals stricken with botulism disease. In addition, collection of human sera must involve screening out HIV and other potentially serious human pathogens. P. J. Schwarz and S. S. Arnon, Western J. Med. 156:197 (1992).!

Infant botulism has been implicated as the cause of mortality in some cases of Sudden Infant Death Syndrome (SIDS, also known as crib death). SIDS is officially recognized as infant death that is sudden and unexpected and that remained unexplained despite complete post-mortem examination. The link of SIDS to infant botulism came when fecal or blood specimens taken at autopsy from SIDS infants were found to contain C. botulinum organisms and/or toxin in 3-4% of cases analyzed. D. R. Peterson et al., Rev. Infect. Dis. 1:630 (1979).! In contrast, only 1 of 160 healthy infants (0.6%) had C. botulinum organisms in the feces and no botulinal toxin. (S. Arnon et al., Lancet, pp. 1273-76, Jun. 17, 1978.)

In developed countries, SIDS is the number one cause of death in children between one month and one year old. (S. Arnon et al., Lancet, pp. 1273-77, Jun. 17, 1978.) More children die from SIDS in the first year than from any other single cause of death in the first fourteen years of life. In the United States, there are 8,000-10,000 SIDS victims annually. Id.

What is needed is an effective therapy against infant botulism that is free of dangerous side effects, is available in large supply at a reasonable price, and can be safely and gently delivered so that prophylactic application to infants is feasible.

Immunization of subjects with toxin preparations has been done in an attempt to induce immunity against botulinal toxins. A C. botulinum vaccine comprising chemically inactivated (i.e., formaldehyde-treated) type A, B,C, D and E toxin is commercially available for human usage. However, this vaccine preparation has several disadvantages. First, the efficacy of this vaccine is variable (in particular, only 78% of recipients produce protective levels of anti-type B antibodies following administration of the primary series). Second, immunization is painful (deep subcutaneous inoculation is required for administration), with adverse reactions being common (moderate to severe local reactions occur in approximately 6% of recipients upon initial injection; this number rises to approximately 11% of individuals who receive booster injections) Informational Brochure for the Pentavalent (ABCDE) Botulinum Toxoid, Centers for Disease Control!. Third, preparation of the vaccine is dangerous as active toxin must be handled by laboratory workers.

What is needed are safe and effective vaccine preparations for administration to those at risk of exposure to C. botulinum toxins.

C. difficile

C. difficile, an organism which gained its name due to difficulties encountered in its isolation, has recently been proven to be an etiologic agent of diarrheal disease. (Sneath et al., p. 1165.). C. difficile is present in the gastrointestinal tract of approximately 3% of healthy adults, and 10-30% of neonates without adverse effect (Swartz, at p. 644); by other estimates, C. difficile is a part of the normal gastrointestinal flora of 2-10% of humans. G. F. Brooks et al., (eds.) "Infections Caused by Anaerobic Bacteria," Jawetz, Melnick, & Adelberg's Medical Microbiology, 19th ed., pp. 257-262, Appleton & Lange, San Mateo, Calif. (1991).! As these organisms are relatively resistant to most commonly used antimicrobials, when a patient is treated with antibiotics, the other members of the normal gastrointestinal flora are suppressed and C. difficile flourishes, producing cytopathic toxins and enterotoxins. It has been found in 25% of cases of moderate diarrhea resulting from treatment with antibiotics, especially the cephalosporins, clindamycin, and ampicillin. M. N. Swartz at 644.!

Importantly, C. difficile is commonly associated with nosocomial infections. The organism is often present in the hospital and nursing home environments and may be carried on the hands and clothing of hospital personnel who care for debilitated and immunocompromised patients. As many of these patients are being treated with antimicrobials or other chemotherapeutic agents, such transmission of C. difficile represents a significant risk factor for disease. (Engelkirk et al., pp. 64-67.)

C. difficile is associated with a range of diarrhetic illness, ranging from diarrhea alone to marked diarrhea and necrosis of the gastrointestinal mucosa with the accumulation of inflammatory cells and fibrin, which forms a pseudomembrane in the affected area. (Brooks et al.) It has been found in over 95% of pseudomembranous enterocolitis cases. (Swartz, at p. 644.) This occasionally fatal disease is characterized by diarrhea, multiple small colonic plaques, and toxic megacolon. (Swartz, at p. 644.) Although stool cultures are sometimes used for diagnosis, diagnosis is best made by detection of the heat labile toxins present in fecal filtrates from patients with enterocolitis due to C. difficile. (Swartz, at p. 644-645; and Brooks et al., at p. 260.) C. difficile toxins are cytotoxic for tissue/cell cultures and cause enterocolitis when injected intracecally into hamsters. (Swartz, at p. 644.)

The enterotoxicity of C. difficile is primarily due to the action of two toxins, designated A and B, each of approximately 300,000 in molecular weight. Both are potent cytotoxins, with toxin A possessing direct enterocytotoxic activity. Lyerly et al., Infect. Immun. 60:4633 (1992).! Unlike toxin A of C. perfringens, an organism rarely associated with antimicrobial-associated diarrhea, the toxin of C. difficile is not a spore coat constituent and is not produced during sporulation. (Swartz, at p. 644.) C. difficile toxin A causes hemorrhage, fluid accumulation and mucosal damage in rabbit ileal loops and appears to increase the uptake of toxin B by the intestinal mucosa. Toxin B does not cause intestinal fluid accumulation, but it is 1000 times more toxic than toxin A to tissue culture cells and causes membrane damage. Although both toxins induce similar cellular effects such as actin disaggregation, differences in cell specificity occurs.

Both toxins are important in disease. Borriello et al., Rev. Infect. Dis., 12(suppl. 2):S185 (1990); Lyerly et al., Infect. Immun., 47:349 (1985); and Rolfe, Infect. Immun., 59:1223 (1990).! Toxin A is thought to act first by binding to brush border receptors, destroying the outer mucosal layer, then allowing toxin B to gain access to the underlying tissue. These steps in pathogenesis would indicate that the production of neutralizing antibodies against toxin A may be sufficient in the prophylactic therapy of CDAD. However, antibodies against toxin B may be a necessary additional component for an effective therapeutic against later stage colonic disease. Indeed, it has been reported that animals require antibodies to both toxin A and toxin B to be completely protected against the disease. Kim and Rolfe, Abstr. Ann. Meet. Am. Soc. Microbiol., 69:62 (1987).!

C. difficile has also been reported to produce other toxins such as an enterotoxin different from toxins A and B Banno et al., Rev. Infect. Dis., 6(Suppl. 1:S11-S20 (1984)!, a low molecular weight toxin Rihn et al., Biochem. Biophys. Res. Comm., 124:690-695 (1984)!, a motility altering factor Justus et al., Gastroenterol., 83:836-843 ( 1982)!, and perhaps other toxins. Regardless, C. difficile gastrointestinal disease is of primary concern.

It is significant that due to its resistance to most commonly used antimicrobials, C. difficile is associated with antimicrobial therapy with virtually all antimicrobial agents (although most commonly ampicillin, clindamycin and cephalosporins). It is also associated with disease in patients undergoing chemotherapy with such compounds as methotrexate, 5-fluorouracil, cyclophosphamide, and doxorubicin. S. M. Finegold et al., Clinical Guide to Anaerobic Infections, pp. 88-89, Star Publishing Co., Belmont, Calif. (1992).!

Treatment of C. difficile disease is problematic, given the high resistance of the organism. Oral metronidazole, bacitracin and vancomycin have been reported to be effective. (Finegold et al., p. 89.) However there are problems associated with treatment utilizing these compounds. Vancomycin is very expensive, some patients are unable to take oral medication, and the relapse rate is high (20-25%), although it may not occur for several weeks. Id.

C. difficile disease would be prevented or treated by neutralizing the effects of these toxins in the gastrointestinal tract. Thus, what is needed is an effective therapy against C. difficile toxin that is free of dangerous side effects, is available in large supply at a reasonable price, and can be safely delivered so that prophylactic application to patients at risk of developing pseudomembranous enterocolitis can be effectively treated.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the reactivity of anti-C. botulinum IgY by Western blot.

FIG. 2 shows the IgY antibody titer to C. botulinum type A toxoid in eggs, measured by ELISA.

FIG. 3 shows the results of C. difficile toxin A neutralization assays.

FIG. 4 shows the results of C. difficile toxin B neutralization assays.

FIG. 5 shows the results of C. difficile toxin B neutralization assays.

FIG. 6 is a restriction map of C. difficile toxin A gene, showing sequences of primers 1-4 (SEQ ID NOS:1-4).

FIG. 7 is a Western blot of C. difficile toxin A reactive protein.

FIG. 8 shows C. difficile toxin A expression constructs.

FIG. 9 shows C. difficile toxin A expression constructs.

FIG. 10 shows the purification of recombinant C. difficile toxin A.

FIG. 11 shows the results of C. difficile toxin A neutralization assays with antibodies reactive to recombinant toxin A.

FIG. 12 shows the results for a C. difficile toxin A neutralization plate.

FIG. 13 shows the results for a C. difficile toxin A neutralization plate.

FIG. 14 shows the results of recombinant C. difficile toxin A neutralization assays.

FIG. 15 shows C. difficile toxin A expression constructs.

FIG. 16 shows a chromatograph plotting absorbance at 280 nm against retention time for a pMA1870-680 IgY PEG preparation.

FIG. 17 shows two recombinant C. difficile toxin B expression constructs.

FIG. 18 shows C. difficile toxin B expression constructs.

FIG. 19 shows C. difficile toxin B expression constructs.

FIG. 20 shows C. difficile toxin B expression constructs.

FIG. 21 is an SDS-PAGE gel showing the purification of recombinant C. difficile toxin B fusion protein.

FIG. 22 is an SDS-PAGE gel showing the purification of two histidine-tagged recombinant C. difficile toxin B proteins.

FIG. 23 shows C. difficile toxin B expression constructs.

FIG. 24 is a Western blot of C. difficile toxin B reactive protein.

FIG. 25 shows C. botulinum type A toxin expression constructs; constructs used to provide C. botulinum or C. difficile sequences are also shown.

FIG. 26 is an SDS-PAGE gel stained with Coomaisse blue showing the purification of recombinant C. botulinum type A toxin fusion proteins.

FIG. 27 shows C. botulinum type A toxin expression constructs; constructs used to provide C. botulinum sequences are also shown. Polyhistidine tags are represented by "HHHHH" (SEQ ID NO:32).

FIG. 28 is an SDS-PAGE gel stained with Coomaisse blue showing the purification of pHisBot protein using the Ni-NTA resin.

FIG. 29 is an SDS-PAGE gel stained with Coomaisse blue showing the expression of pHisBot protein in BL21(DE3) and BL21(DE3)pLysS host cells.

FIG. 30 is an SDS-PAGE gel stained with Coomaisse blue showing the purification of pHisBot protein using a batch absorption procedure.

FIG. 31 shows C. difficile toxin A expression constructs.

FIG. 32 shows an SDS-PAGE gel stained with Coomaisse blue and a Western blot showing the expression of the pUC1960-2680 in E. coli host cells.

FIG. 33 shows an SDS-PAGE gel stained with Coomaisse blue and a Western blot showing the expression of the several recombinant C. difficile toxin A fusion proteins in E. coli host cells.

FIG. 34 is an SDS-PAGE gel stained with Coomaisse blue showing the purification of recombinant C. difficile toxin A and B fusion proteins.

FIG. 35 shows the results of a prophylactic treatment study in hamsters.

FIG. 36 shows the results of a therapeutic treatment study in hamsters.

FIG. 37 shows the results of a therapeutic treatment study in hamsters.

FIG. 38 shows the results of a therapeutic treatment study in hamsters.

FIG. 39 shows the results of administration of vancomycin to hamsters having an established C. difficile infection.

FIG. 40 shows the results of an ELISA analysis of IgY isolated from hens immunized with the recombinant C. difficile toxin A protein pMA1870-2680 and four different adjuvants.

FIG. 41 shows the results of an ELISA analysis of IgY isolated from hens immunized with the recombinant C. difficile toxin A protein pPA1870-2680(N/C) and four different adjuvants.

FIG. 42 shows dissolution profiles for Aquateric-coated IgY.

FIG. 43 shows dissolution profiles for Eugragit®-coated IgY.

FIG. 44 shows the results of an ELISA analysis of IgY isolated from hamsters vaccinated with recombinant C. difficile toxin A proteins.

FIG. 45 shows the results of an ELISA analysis of IgY isolated from hamsters vaccinated with recombinant C. difficile toxin A and B proteins; reactivity to recombinant C. difficile toxin A is shown.

FIG. 46 shows the results of an ELISA analysis of IgY isolated from hamsters vaccinated with recombinant C. difficile toxin A and B proteins; reactivity to recombinant C. difficile toxin B is shown.

FIG. 47 results of a therapeutic treatment study in hamsters.

FIG. 48 results of a therapeutic treatment study in diarrhetic hamsters.

FIG. 49 results of a therapeutic treatment study in hamsters.

FIG. 50 shows a Western blot showing C. difficile toxin A levels in culture supernatant, column flow through and column eluate from an affinity purification column.

FIG. 51 shows a Western blot showing C. difficile toxin A levels in culture supernatant, column flow through and column eluate from an affinity purification column.

FIG. 52 is an native PAGE gel stained with Coomaisse blue showing C. difficile toxin B levels in liquid culture supernatant.

FIG. 53 is an native PAGE gel stained with Coomaisse blue and a Western blot showing C. difficile toxin B levels in dialysis bag cultures.

FIG. 54 is an native PAGE gel stained with Coomaisse blue and a Western blot showing C. difficile toxin B levels in a commercial toxin B preparation and column flow through and column eluate from an affinity purification column.

DEFINITIONS

To facilitate understanding of the invention, a number of terms are defined below.

As used herein, the term "neutralizing" is used in reference to antitoxins, particularly antitoxins comprising antibodies, which have the ability to prevent the pathological actions of the toxin against which the antitoxin is directed.

As used herein, the term "overproducing" is used in reference to the production of clostridial toxin polypeptides in a host cell and indicates that the host cell is producing more of the clostridial toxin by value of the introduction of nucleic acid sequences encoding said clostridial toxin polypeptide than would be expressed by said host cell absent the introduction of said nucleic acid sequences. To allow ease of purification of toxin polypeptides produced in a host cell it is preferred that the host cell express or overproduce said toxin polypeptide at a level greater than 1 mg/liter of host cell culture.

As used herein, the term "fusion protein" refers to a chimeric protein containing the protein of interest (i.e., C. difficile toxin A or B and fragments thereof) joined to an exogenous protein fragment (the fusion partner which consists of a non-toxin protein). The fusion partner may enhance solubility of the C. difficile protein as expressed in a host cell, may provide an affinity tag to allow purification of the recombinant fusion protein from the host cell or culture supernatant, or both. If desired, the fusion protein may be removed from the protein of interest (i.e., toxin protein or fragments thereof) prior to immunization by a variety of enzymatic or chemical means known to the art.

As used herein the term "non-toxin protein" or "non-toxin protein sequence" refers to that portion of a fusion protein which comprises a protein or protein sequence which is not derived from a bacterial toxin protein.

The term "protein of interest" as used herein refers to the protein whose expression is desired within the fusion protein. In a fusion protein the protein of interest will be joined or fused with another protein or protein domain, the fusion partner, to allow for enhanced stability of the protein of interest and/or ease of purification of the fusion protein.

As used herein, the term "maltose binding protein" refers to the maltose binding protein of E. coli. A portion of the maltose binding protein may be added to a protein of interest to generate a fusion protein; a portion of the maltose binding protein may merely enhance the solubility of the resulting fusion protein when expressed in a bacterial host. On the other hand, a portion of the maltose binding protein may allow affinity purification of the fusion protein on an amylose resin.

As used herein, the term "poly-histidine tract" when used in reference to a fusion protein refers to the presence of two to ten histidine residues at either the amino- or carboxy-terminus or both termini of a protein of interest or a fusion partner. A poly-histidine tract of six to ten residues is preferred. The poly-histidine tract is also defined functionally as being a number of consecutive histidine residues added to the protein of interest which allows the affinity purification of the resulting fusion protein on a nickel-chelate column.

The term "thioredoxin protein" when used in reference to a fusion protein refers to a the thioredoxin protein of E. coli. It is noted that the invention is not limited by the source of the thioredoxin protein, while the E. coli thioredoxin protein is particularly preferred, thioredoxin proteins may be obtained from several sources. A portion of the thioredoxin protein may be added to a protein of interest to generate a fusion protein; a portion of the thioredoxin protein may enhance the solubility of the resulting fusion protein when expressed in a bacterial host.

As used herein, the term "purified" or "to purify" refers to the removal of contaminants from a sample. For example, antitoxins are purified by removal of contaminating non-immunoglobulin proteins; they are also purified by the removal of immunoglobulin that does not bind toxin. The removal of non-immunoglobulin proteins and/or the removal of immunoglobulins that do not bind toxin results in an increase in the percent of toxin-reactive immunoglobulins in the sample. The purification of antitoxin may be accomplished by a variety of means including the extraction and precipitation of avian antitoxin from eggs using polyethylene glycol. Purification of anticlostridal antitoxin may also be accomplished by affinity chromatography on a resin comprising a portion of a clostridial toxin protein. In another example, recombinant toxin polypeptides are expressed in bacterial host cells and the toxin polypeptides are purified by the removal of host cell proteins; the percent of recombinant toxin polypeptides is thereby increased in the sample. Additionally, the recombinant toxin polypeptides are purified by the removal of host cell components such as lipopolysaccharide (e.g., endotoxin).

The term "recombinant DNA molecule" as used herein refers to a DNA molecule which is comprised of segments of DNA joined together by means of molecular biological techniques.

The term "recombinant protein" or "recombinant polypeptide" as used herein refers to a protein molecule which is expressed from a recombinant DNA molecule.

The term "native protein" as used herein refers to a protein which is isolated from a natural source as opposed to the production of a protein by recombinant means.

As used herein the term "portion" when in reference to a protein (as in "a portion of a given protein") refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.

As used herein "soluble" when in reference to a protein produced by recombinant DNA technology in a host cell is a protein which exists in solution in the cytoplasm of the host cell; if the protein contains a signal sequence the soluble protein is exported to the periplasmic space in bacteria hosts and is secreted into the culture medium in eucaryotic cells capable of secretion or by bacterial host possessing the appropriate genes (i.e., the kil gene). In contrast, an insoluble protein is one which exists in denatured form inside cytoplasmic granules (called an inclusion bodies) in the host cell. High level expression (i.e., greater than 10-20 mg recombinant protein/liter of bacterial culture) of recombinant proteins often results in the expressed protein being found in inclusion bodies in the bacterial host cells. A soluble protein is a protein which is not found in an inclusion body inside the host cell or is found both in the cytoplasm and in inclusion bodies and in this case the protein may be present at high or low levels in the cytoplasm.

A distinction is drawn between a soluble protein (i.e., a protein which when expressed in a host cell is produced in a soluble form) and a "solubilized" protein. An insoluble recombinant protein found inside an inclusion body may be solubilized (i.e., rendered into a soluble form) by treating purified inclusion bodies with denaturants such as guanidine hydrochloride, urea or sodium dodecyl sulfate (SDS). These denaturants must then be removed from the solubilized protein preparation to allow the recovered protein to renature (refold). Not all proteins will refold into an active conformation after solubilization in a denaturant and removal of the denaturant. Many proteins precipitate upon removal of the denaturant. SDS may be used to solubilize inclusion bodies and will maintain the proteins in solution at low concentration. However, dialysis will not always remove all of the SDS (SDS can form micelles which do not dialyze out); therefore, SDS-solubilized inclusion body protein is soluble but not refolded.

A distinction is drawn between proteins which are soluble (i.e., dissolved) in a solution devoid of significant amounts of ionic detergents (e.g., SDS) or denaturants (e.g., urea, guanidine hydrochloride) and proteins which exist as a suspension of insoluble protein molecules dispersed within the solution. A soluble protein will not be removed from a solution containing the protein by centrifugation using conditions sufficient to remove bacteria present in a liquid medium (i.e., centrifugation at 5,000×g for 4-5 minutes). For example, to test whether two proteins, protein A and protein B, are soluble in solution, the two proteins are placed into a solution selected from the group consisting of PBS-NaCl (PBS containing 0.5M NaCl), PBS-NaCl containing 0.2% Tween 20, PBS, PBS containing 0.2% Tween 20, PBS-C (PBS containing 2 mM CaCl₂), PBS-C containing either 0.1 or 0.5% Tween 20, PBS-C containing either 0.1 or 0.5% NP-40, PBS-C containing either 0.1 or 0.5% Triton X-100, PBS-C containing 0.1% sodium deoxycholate. The mixture containing proteins A and B is then centrifuged at 5000×g for 5 minutes. The supernatant and pellet formed by centrifugation are then assayed for the presence of protein A and B. If protein A is found in the supernatant and not in the pellet except for minor amounts (i.e., less than 10%) as a result of trapping!, protein is said to be soluble in the solution tested. If the majority of protein B is found in the pellet (i.e., greater than 90%), then protein B is said to exist as a suspension in the solution tested.

As used herein, the term "therapeutic amount" refers to that amount of antitoxin required to neutralize the pathologic effects of one or more clostridial toxins in a subject.

The term "therapeutic mixture" when used in reference to a mixture of antitoxins refers to that amount of antitoxin required neutralize the pathologic effects of one or more clostridial toxins in a subject.

The term "therapeutic vaccine" when used in reference to a vaccine comprising one or more recombinant clostridial toxin fusion proteins means that the vaccine contains an immunologically-effective amount of the fusion proteins (i.e., the immunogens.

As used herein the term "immunogenically-effective amount" refers to that amount of an immunogen required to invoke the production of protective levels of antibodies in a host (i.e., a subject) upon vaccination.

The term "pyrogen" as used herein refers to a fever-producing substance. Pyrogens may be endogenous to the host (e.g., prostaglandins) or may be exogenous compounds (e.g., bacterial endo- and exotoxins, nonbacterial compounds such as antigens and certain steroid compounds, etc.). The presence of pyrogen in a pharmaceutical solution may be detected using the U.S. Pharmacopeia (USP) rabbit fever test (United States Pharmacopeia, Vol. XXII (1990) United States Pharmacopeial Convention, Rockville, Md., p. 151).

The term "endotoxin" as used herein refers to the high molecular weight complexes associated with the outer membrane of gram-negative bacteria. Unpurified endotoxin contains lipids, proteins and carbohydrates. Highly purified endotoxin does not contain protein and is referred to as lipopolysaccharide (LPS). Because unpurified endotoxin is of concern in the production of pharmaceutical compounds (e.g., proteins produced in E. coli using recombinant DNA technology), the term endotoxin as used herein refers to unpurified endotoxin. Bacterial endotoxin is a well known pyrogen.

As used herein, the term "endotoxin-free" when used in reference to a composition to be administered parenterally (with the exception of intrathecal administration) to a host means that the dose to be delivered contains less than 5 EU/kg body weight FDA Guidelines for Parenteral Drugs (December 1987)!. Assuming a weight of 70 kg for an adult human, the dose must contain less than 350 EU to meet FDA Guidelines for parenteral administration. Endotoxin levels are measured herein using the Limulus Amebocyte Lysate (LAL) test (Limulus Amebocyte Lysate Pyrochrome™, Associates of Cape Cod, Inc. Woods Hole, Mass.). To measure endotoxin levels in preparations of recombinant proteins, 0.5 ml of a solution comprising 0.5 mg of purified recombinant protein in 50 mM NaPO₄, pH 7.0, 0.3M NaCl and 10% glycerol is used in the LAL assay according to the manufacturer's instructions for the endpoint chromogenic without diazo-coupling method. Compositions containing less than or equal to 450 endotoxin units (EU)/mg of purified recombinant protein are herein defined as "substantially endotoxin-free." Typically, administration of bacterial toxins or toxoids to adult humans for the purpose of vaccination involves doses of about 10-500 μg protein/dose. Therefore, administration of 10-500 μg of a purified recombinant protein to a 70 kg human, wherein said purified recombinant protein preparation contains 450 EU/mg protein, results in the introduction of only 4.5 to 225 EU (i.e., 1.3 to 64.5% of the maximum allowable endotoxin burden per parenteral dose).

The LAL test is accepted by the U.S. FDA as a means of detecting bacterial endotoxins (21 C.F.R. §§ 660.100-105). Studies have shown that the LAL test is equivalent or superior to the USP rabbit pyrogen test for the detection of endotoxin and thus the LAL test can be used as a surrogate for pyrogenicity studies in animals F. C. Perason, Pyrogens: endotoxins, LAL testing and depyrogenation, Marcel Dekker, New York (1985), pp.150-155!. The FDA Bureau of Biologics accepts the LAL assay in place of the USP rabbit pyrogen test so long as the LAL assay utilized is shown to be as sensitive as, or more sensitive as the rabbit test Fed. Reg., 38, 26130 (1980)!.

The term "monovalent" when used in reference to a clostridial vaccine refers to a vaccine which is capable of provoking an immune response in a host (i.e., a subject) animal directed against a single type of clostridial toxin. For example, if immunization of a host with C. difficile type A toxin vaccine induces antibodies in the immunized host which protect against a challenge with type A toxin but not against challenge with type B toxin, then the type A vaccine is said to be monovalent. In contrast, a "multivalent" vaccine provokes an immune response in a host animal directed against several (i.e., more than one) clostridial toxins. For example, if immunization of a host with a vaccine comprising C. difficile type A and B toxins induces the production of antibodies which protect the host against a challenge with both type A and B toxin, the vaccine is said to be multivalent (in particular, this hypothetical vaccine is bivalent).

As used herein, the terms "aggregate" and "aggregation" refer to the production of clumps, groupings, or masses of materials. It is not intended that the terms be limited to a particular type of clumping. Rather, it is intended that the term be used in its broadest sense to encompass any situation where multiple items are brought together into close contact. Thus, the term encompasses agglutination of any type (including, but not limited to latex agglutination, hemagglutination, or any other method in which an immunological reaction is used to produce agglutination). The terms also apply to non-immunological methods, and also encompass non-specific relationships between multiple components; all that is required is that the individual components be clumped together.

The term "subject" when used in reference to administration of compositions comprising antitoxins or vaccines refers to the recipient animal to whom said antitoxins or vaccines are administered. The subject may be any animal, including mammals and more particularly, humans, in which it is desirable to administer said compositions. The subject may have been previously exposed to one or more C. difficile toxins prior to administration of said compositions (this constitutes therapeutic administration to the subject). Alternatively, the subject may not have been previously exposed to C. difficile toxins prior to administration of said compositions (this constitutes prophylactic administration to the subject).

The term "sample" in the present specification and claims is used in its broadest sense. On the one hand it is meant to include a specimen or culture (e.g., microbiological cultures). On the other hand, it is meant to include both biological and environmental samples.

Biological samples may be animal, including human, fluid, solid (e.g., stool) or tissue, as well as liquid and solid food and feed products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Biological samples may be obtained from all of the various families of domestic animals, as well as eral or wild animals, including, but not limited to, such animals as ungulates, bear, fish, lagamorphs, rodents, etc.

Environmental samples include environmental material such as surface matter, soil, water and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention.

As used herein, the term "culture" is used in reference to the in vivo or in vitro growth of organisms, including, but not limited to bacteria. It is intended that the term encompass any form of microbial culture. It is intended that the term encompass the propagation of micoorganisms or other living cells in media and in an environment that is conducive to their growth. Such cultures may be grown in any format, including but not limited to agar plates, broths, and semi-solid media, and may be grown in any environment suitable for the organisms cultured (i.e., aerobic, anaerobic, microaerophilic, etc.).

As used herein, the term "supernatant" is used in reference to any liquid or fluid solution. This liquid or fluid may or may not contain soluble particles such as proteins (e.g., antibodies or toxin molecules). The term encompasses any liquid lying above precipitated insoluble material, as well as liquids such as liquid culture media collected from a microbial or cell culture. It also encompasses the liquid portion of a sample which has been centrifuged to separate insoluble particles which are incapable of remaining in solution during centrifugation, from particles which are capable of remaining in solution during centrifugation. However, it is not intended that the term be limited to the situation in which centrifugation is utilized.

The term "protective level", when used in reference to the level of antibodies induced upon immunization of the host with an immunogen which comprises a bacterial toxin, means a level of circulating antibodies sufficient to protect the host from challenge with a lethal dose of the toxin.

As used herein the terms "protein" and "polypeptide" refer to compounds comprising amino acids joined via peptide bonds and are used interchangeably.

The term "toxin" when used in reference to toxins produced by members (i.e., species and strains) of the genus Clostridium refers to the proteins which are poisonous to tissue(s). For example, the toxins produced by C. difficile are poisonous to intestinal tissues; the toxins produced by C. botulinum are poisonous to nerve tissue.

The terms "encapsulation" or "encapsulating" refers to the covering of a solid (e.g., lyophilized) form of antitoxin. The covering may comprise an enteric coating or a capsule. The terms "enteric coating" or "enteric film" are used interchangeably and refer to a material or compound which is resistant to acid pH (i.e., an acid-resistant compound), such as that found in the stomach. An enteric coating when applied to a solid inhibits the dissolution of the solid in the stomach.

Standard techniques are known to the art for the encapsulation of solid compositions. These techniques include microencapsulation of a solid composition wherein an enteric coating is applied to the solid composition. The coated material may be delivered orally to a subject by suspending the microencapsulated particles in pharmaceutical suspension solutions known to the art.

When a solid antitoxin is to be encapsulated using an enteric coating, the enteric coating may be applied using a one step coating process in which the enteric film is directly applied to the solid antitoxin; the coated antitoxin is said to be overcoated with the enteric film. Alternatively, a two step coating process may be employed wherein the solid antitoxin is first used to overcoat a non-pariel (i.e., a sugar particle of about 40-60 mesh size) and then the antitoxin-coated non-pariel is overcoated with the enteric film. Desirable enteric coatings for the delivery of antitoxins include polymethacrylates such as Eudragit® L30D (Rohm Tech, Inc.)

Solid antitoxin may formulated for oral delivery by insertion of the desired quantity of antitoxin into a capsule; the capsule would preferable have the characteristic of being resistant to dissolution in the stomach and being capable of dissolving in the intestines. Numerous suitable capsule formulations are available to the art; in addition standard techniques are available for the filling of capsules including the use of inert filler materials to provide sufficient bulk of the filling of a capsule with a therapeutic composition in a solid form. In addition to the use of microencapsulated antitoxin and antitoxin contained within a capsule, the solid antitoxin may be delivered orally in tablet or pill form. The solid antitoxin may be combined with inert materials to provide sufficient bulk for the pressing of the tablet or pill. Once formed, the tablet or pill may then be coated with an enteric film to prevent dissolution in the stomach and to enhance dissolution in the intestines.

The term "oral administration" refers to the delivery of a composition, such as a composition comprising antitoxin, via the mouth.

The term "parenteral administration" refers to the delivery of a composition, such as a composition comprising an antitoxin or vaccine, by a route other than through the gastrointestinal tract (e.g., oral delivery) or the lungs. In particular, parenteral administration may be via intravenous, subcutaneous, intramuscular or intramedullary (i.e., intrathecal) injection.

The terms "symptoms" and "symptoms of intoxication" when used in reference to a subject exposed to or at risk of exposure to C. difficile toxins refers to presence of any of the following phenomenon: diarrhea, enterocolitis, pseudomembranous colitis, hemorrhage, ulceration and/or inflammation of the intestinal mucosa, cecitis (i.e., inflammation of the cecum).

As used herein, the term "ceases to exhibit symptoms" refers to the situation in which a subject has stopped exhibiting the signs and/or symptoms associated with C. difficile disease and/or infection.

The term "substantial elimination" of the symptoms of intoxication with C. difficile disease means that in subject exposed to and suffering from the symptoms of intoxication, the symptoms are abated, attenuated or eliminated. For example, if an intoxicated subject presents with severe diarrhea (i.e., voluminous, watery diarrhea), a return to an at least loosely formed stool would constitute a substantial elimination of this symptom.

The term "beyond the treatment period" when used in reference to a method of treating a subject exposed to a C. difficile toxin means a period of time following the cessation of administration of a therapeutic compound (e.g., antitoxin) to the subject for at least 7 days and more preferably at least 14 days. A therapeutic compound which results in the substantial elimination of the symptoms of intoxication beyond the treatment period will prevent the reappearance (when symptoms are eliminated) or the increase in severity (when symptoms are abated) of these symptoms for at least 7 days following the withdrawal of administration of the therapeutic compound. In other words, no relapse (i.e., reappearance or increase in severity) of the symptoms is seen in the majority i.e., a statistically significant number (e.g., 75%)! of subjects for a period of at least 7 days following the cessation of therapy.

In contrast to the antitoxins of the present invention, existing therapeutic compounds for established C. difficile infections i.e., antibiotics such as vancomycin or metronidazole or bovine IgG concentrate from cows immunized with C. difficile toxoids A and B Lyerly et al. (1991) Infect. Immun. 59:2215! do not prevent relapse in a significant number of treated subjects. For example, about 25% of humans and up to 100% of hamsters suffering from C. difficile associated disease treated with either vancomycin or metronidazole relapse (i.e., symptoms of intoxication reappear).

Hamsters administered bovine IgG concentrate (BIC) from cows immunized with C. difficile toxoids A and B prior to infection with C. difficile (i.e., prophylactic treatment) invariably relapse (i.e., diarrheas returns) and die when the BIC is withdrawn Lyerly et al. (1991), supra!. No therapeutic effect is observed when hamsters having established C. difficile infections are treated with the BIC (i.e., the administration of the BIC does not eliminate the diarrhea or prevent death) Lyerly et al. (1991), supra!.

In contrast, the antitoxins of the present invention, when used to treat established C. difficile infection (therapeutic regimen), substantially eliminate the symptoms of intoxication, including diarrhea and prevent death. The majority of animals treated with the anti-C. difficile toxin proteins do not relapse and remain healthy following cessation of antitoxin therapy for a period of at least 14 days the animals remain healthy for long periods of time (e.g., about 5 months)!.

SUMMARY OF THE INVENTION

The present invention provides compositions comprising an avian neutralizing antitoxin directed against a portion of C. difficile toxin A and a portion of C. difficile toxin B. The antitoxins find use in humans and other animals exposed to or at risk of exposure to C. difficile. In one embodiment, the component of the avian neutralizing antitoxin directed against a portion C. difficile toxin A is directed against a first fusion protein comprising a portion of C. difficile toxin A and a second fusion protein comprising a portion of C. difficile toxin B. In yet another embodiment, both first and second fusion proteins further comprise at least one non-toxin protein sequence. In a still further embodiment, the antitoxin is directed against a portion of C. difficile toxin A comprising a portion of SEQ ID NO:6. In another embodiment, the antitoxin is directed against a portion of C. difficile toxin A, wherein the portion of SEQ ID NO:6 comprises a sequence selected from the group comprising SEQ ID NOS:7, 8 and 29. In yet another embodiment, the first and second fusion proteins comprise at least one non-toxin protein sequence. It is not intended that the present invention be limited by the nature of the non-toxin protein sequence. In one embodiment, the non-toxin protein sequence comprises a poly-histidine tract. In yet another embodiment, the non-toxin protein sequence comprises the maltose binding protein. In yet another embodiment, the non-toxin protein sequence comprises a thioredoxin protein. In a still further embodiment, the antitoxin is directed against a portion of C. difficile toxin B comprising a portion of SEQ ID NO:10. In another embodiment, the antitoxin is directed against a portion of C. difficile toxin B, wherein the portion of SEQ ID NO:10 comprises a sequence selected from the group comprising SEQ ID NOS:20, 21 and 30. In still another embodiment, the compositions comprising the avian antitoxins further comprise an enteric coating.

The invention also contemplates a method of treatment comprising: a) providing: i) a subject, ii) a first avian neutralizing antitoxin directed against a portion of Clostridium difficile toxin A sequence SEQ ID NO:6, and iii) a second avian neutralizing antitoxin directed against a portion of Clostridium difficile toxin B sequence SEQ ID NO:10; b) mixing the first and second antitoxins to create a therapeutic mixture; and c) administering the therapeutic mixture to a subject for a treatment period. The invention further contemplates a method of treatment which further comprises the step of, prior to step c), processing the therapeutic mixture to improve its enteric stability. In a preferred embodiment, this treating comprises encapsulating the antitoxins of the therapeutic mixture. In a particularly preferred embodiment the encapsulating step comprises overcoating the antitoxins in the therapeutic mixture with an enteric film.

The invention further contemplates the method of treatment wherein the subject has been exposed to at least one Clostridium difficile toxin prior to administration of antitoxin. In one embodiment, the exposed subject is suffering from the symptoms of intoxication and administering antitoxin results in the substantial elimination of symptoms beyond the treatment period. In another embodiment, the symptoms of intoxication comprise diarrhea.

The invention also contemplates the method of treatment wherein the subject has not been exposed to Clostridium difficile toxin prior to administration of antitoxin.

In one embodiment, the method of treatment provides a first avian antitoxin directed against a portion of Clostridium difficile toxin A comprising a protein sequence selected from the group comprising SEQ ID NOS:7, 8 and 29. In another embodiment, the method of treatment provides a second avian antitoxin directed against a portion of Clostridium difficile toxin B comprising a protein sequence selected from the group comprising SEQ ID NOS:11, 12, 20, 21 and 30.

The method of treatment is not limited by the method of administration of the antitoxin. In one embodiment, the method of treatment comprises administration of the antitoxins by oral administration. In another embodiment, the method of treatment comprises administration of the antitoxins by parenteral administration.

The invention further contemplates a method of vaccinating a subject to produce neutralizing antitoxin directed against C. difficile toxin comprising: a) providing in any order: i) a subject, ii) a first purified soluble and substantially endotoxin-free protein comprising a portion of Clostridium difficile toxin A sequence SEQ ID NO:6, and iii) a second purified soluble and substantially endotoxin-free protein comprising a portion of Clostridium difficile toxin B sequence SEQ ID NO:10; B) mixing the first and second proteins to create a therapeutic vaccine; and c) vaccinating the subject with the therapeutic vaccine so as to generate neutralizing antitoxin. The method of vaccination is not limited by the nature or species of the subject. In one embodiment the subject is a bird. In another embodiment the subject is a mammal. In yet another embodiment the subject is a human. In a still further embodiment, the method of vaccination the first and second toxin proteins further comprise at least one non-toxin protein sequence. The invention is not limited by the nature of the non-toxin protein sequence. In one embodiment, the non-toxin protein sequence comprises a poly-histidine tract. In another embodiment, the non-toxin protein sequence comprises the maltose binding protein. In yet another embodiment, the non-toxin protein sequence comprises a thioredoxin protein.

In one embodiment, the method of vaccinating uses a first purified and substantially endotoxin-free protein comprising SEQ ID NO:29. In another embodiment, the method of vaccinating uses a second purified and substantially endotoxin-free protein comprising SEQ ID NO:30.

The invention further provides a fusion protein comprising at least one non-toxin protein sequence and a portion of the Clostridium difficile toxin A sequence consisting of SEQ ID NO:29. In one embodiment, the non-toxin protein sequence comprises a thioredoxin protein. In yet another embodiment, the non-toxin protein sequence further comprises a poly-histidine tract.

The present invention provides a method for the detection of Clostridium difficile antigens in a sample, comprising providing, in any order a sample suspected of containing Clostridium difficile antigens, solid support conjugates comprising antibodies reactive with Clostridium difficile antigens bound to a solid support; mixing the sample and solid support conjugates under conditions such that the conjugates are capable of binding to Clostridium difficile antigens; and detecting binding. In one embodiment, the antibodies reactive with Clostridium difficile antigens are avian antibodies. In a preferred embodiment, the avian antibodies reacts with Toxin A of Clostridium difficile. In a particularly preferred embodiment, the avian antibodies reacts with the A-6 interval of Toxin A. In an alternative preferred embodiment the avian antibodies react with Toxin B of Clostridium difficile. In another preferred embodiment, the avian antibodies react with the B-3 interval of Toxin B. In yet another preferred embodiment, the avian antibodies react with Toxin A and Toxin B. It is also contemplated that the solid support used in the method comprises polystyrene particles. In one preferred embodiment, the mixing of step results in the formation of visible aggregates. In a preferred embodiment, the sample is human feces.

In an alternative embodiment, the present invention comprises a method of treatment comprising providing a subject exposed to Clostridium difficile exhibiting symptoms comprising diarrhea, and antibody reactive with Clostridium difficile, wherein the antibody is present in a therapeutic amount that is administrable, and administering the antibody to the subject under conditions such that the subject ceases to exhibit symptoms and treatment can be terminated. In a particularly preferred embodiment, the subject exhibits long-term survival beyond the treatment period. In one preferred embodiment, the antibodies reactive with Clostridium difficile antigens are avian antibodies. It is contemplated that the antibodies will be reactive against various moieties or antigens, including, but not limited to Toxin A of Clostridium difficile, the A-6 interval of Toxin A, Toxin B of Clostridium difficile, the B-3 interval of Toxin B, and a combination of Toxin A and Toxin B.

The present invention also provides a method of purifying Clostridium difficile toxins from a culture, comprising providing in any order, a culture comprising Clostridium difficile organisms and a supernatant comprising toxins in solution, antibodies reactive with Clostridium difficile toxins immobiled on a solid support, collecting the supernatant from the culture comprising toxins, adding the supernatant to immobilized antibody under conditions such that antibodies are capable of binding to the toxins, eluting the toxins from the immobilized antibodies; and detecting any eluted toxins. In one preferred embodiment, the antibodies reactive with Clostridium difficile antigens are avian antibodies. It is contemplated that various antibodies will be used in this method, including antibodies reactive against various antigens or moieties, including, but not limited to Toxin A of Clostridium difficile, the A-6 interval of Toxin A, Toxin B of Clostridium difficile, the B-3 interval of Toxin B, and a combination of Toxin A and Toxin B.

DESCRIPTION OF THE INVENTION

The present invention contemplates vaccinating humans and other animals polypeptides derived from C. botulinum neurotoxin which are substantially endotoxin-free. These botulinal peptides are also useful for the production of antitoxin. Anti-botulinal toxin antitoxin is useful for the treatment of patients effected by or at risk of symptoms due to the action of C. botulinum toxins. The organisms, toxins and individual steps of the present invention are described separately below.

I. Clostridium Species, Clostridial Diseases And Associated Toxins

A preferred embodiment of the method of the present invention is directed toward obtaining antibodies against Clostridium species, their toxins, enzymes or other metabolic by-products, cell wall components, or synthetic or recombinant versions of any of these compounds. It is contemplated that these antibodies will be produced by immunization of humans or other animals. It is not intended that the present invention be limited to any particular toxin or any species of organism. In one embodiment,

                  TABLE 2                                                          ______________________________________                                         Clostridial Toxins                                                             Organism  Toxins and Disease-Associated Antigens                               ______________________________________                                         C. botulinum                                                                             A, B, C.sub.1, C.sub.2, D, E, F, G                                   C. butyricum                                                                             Neuraminidase                                                        C. difficile                                                                             A, B, Enterotoxin (not A nor B), Motility Altering                             Factor, Low Molecular Weight Toxin, Others                           C. perfringens                                                                           α, β, ε, ι, γ, δ, ν,                    θ, κ, λ, μ, υ                          C. sordelli/                                                                             HT, LT, α, β, γ                                     C. bifermentans                                                                C. novyi  α, β, γ, δ, ε, ζ, ν,                    θ                                                              C. septicum                                                                              α, β, γ, δ                                    C. histolyticum                                                                          α, β, γ, δ, ε plus additional                   enzymes                                                              C. chauvoei                                                                              α, β, γ, δ                                    ______________________________________                                    

toxins from all Clostridium species are contemplated as immunogens. Examples of these toxins include the neuraminidase toxin of C. butyricum, C. sordellii toxins HT and LT, toxins A, B, C, D, E, F, and G of C. botulinum and the numerous C. perfringens toxins. In one preferred embodiment, toxins A and B of C. difficile are contemplated as immunogens. Table 2 above lists various Clostridium species, their toxins and some antigens associated with disease.

It is not intended that antibodies produced against one toxin will only be used against that toxin. It is contemplated that antibodies directed against one toxin (e.g., C. perfringens type A enterotoxin) may be used as an effective therapeutic against one or more toxin(s) produced by other members of the genus Clostridium or other toxin producing organisms (e.g., Bacillus cereus, Staphylococcus aureus, Streptococcus mutans, Acinetobacter calcoaceticus, Pseudomonas aeruginosa, other Pseudomonas species, etc.). It is further contemplated that antibodies directed against the portion of the toxin which binds to mammalian membranes (e.g., C. perfringens enterotoxin A) can also be used against other organisms. It is contemplated that these membrane binding domains are produced synthetically and used as immunogens.

II. Obtaining Antibodies In Non-Mammals

A preferred embodiment of the method of the present invention for obtaining antibodies involves immunization. However, it is also contemplated that antibodies could be obtained from non-mammals without immunization. In the case where no immunization is contemplated, the present invention may use non-mammals with preexisting antibodies to toxins as well as non-mammals that have antibodies to whole organisms by virtue of reactions with the administered antigen. An example of the latter involves immunization with synthetic peptides or recombinant proteins sharing epitopes with whole organism components.

In a preferred embodiment, the method of the present invention contemplates immunizing non-mammals with bacterial toxin(s). It is not intended that the present invention be limited to any particular toxin. In one embodiment, toxin from all clostridial bacteria sources (see Table 2) are contemplated as immunogens. Examples of these toxins are C. butyricum neuraminidase toxin, toxins A, B, C, D, E, F, and G from C. botulinum, C. perfringens toxins α, β, ε, and ι, C. sordellii toxins HT and LT. In a preferred embodiment, C. difficile toxins A and B are contemplated as immunogens.

A particularly preferred embodiment involves the use of bacterial toxin protein or fragments of toxin proteins produced by molecular biological means (i.e., recombinant toxin proteins). In a preferred embodiment, the immunogen comprises interval 6 of C. difficile toxin A produced by recombinant DNA technology. In yet another preferred embodiment, the immunogen comprises interval 3 of C. difficile toxin B produced by recombinant DNA technology. The recombinant C. difficile toxin proteins may be used as immunogens separately or in combination to produce antibodies specific for either C. difficile toxin A, C. difficile toxin B or both C. difficile toxins A and B. Specifically, the recombinant C. difficile toxins A and B proteins may be mixed together and used as a single immunogen. Alternatively, C. difficile toxin A proteins may be used separately as an immunogen in a first subject. Similarly, C. difficile toxin B proteins may be used separately as an immunogen in a second subject. The antitoxin produced by separate immunization of two separate subjects with C. difficile toxin A proteins or C. difficile toxin B proteins may be combined to yield an antitoxin directed against both C. difficile toxins A and B.

The recombinant C. difficile toxin proteins provided herein enables the production of antibodies which are specific for a single C. difficile toxin (i.e., mono-specific antibodies). This is in contrast to the biochemical purification of C. difficile toxin A from natural sources results invariably in the isolation of a toxin A preparation contaminated with immunologically significant amounts of toxin B; similarly the biochemical purification of C. difficile toxin B from natural sources results in the isolation of a toxin B preparation contaminated with immunologically significant amounts of toxin A. Because, these preparations of non-recombinant toxin A and or toxin B are cross-contaminated with either toxin B or A, immunization of an animal will result in the production of polyclonal antibodies reactive against both toxins A and B.

As discussed below in section VI, accurate detection of the presence of C. difficile toxin A and/or B in a sample requires the availability of both pure preparations of toxin A and B and the availability of mono-specific antibodies. The use of recombinant C. difficile toxin proteins thus allows for the production of a polyclonal antibody preparation that can be used for accurate detection of individual C. difficile toxins as well as C. difficile organisms.

When immunization is used, the preferred non-mammal is from the class Aves. All birds are contemplated (e.g., duck, ostrich, emu, turkey, etc.). A preferred bird is a chicken. Importantly, chicken antibody does not fix mammalian complement. See H. N. Benson et al., J. Immunol. 87:616 (1961).! Thus, chicken antibody will normally not cause a complement-dependent reaction. A. A. Benedict and K. Yamaga, "Immunoglobulins and Antibody Production in Avian Species," in Comparative Immunology (J. J. Marchaloni, ed.), pp. 335-375, Blackwell, Oxford (1966).! Thus, the preferred antitoxins of the present invention will not exhibit complement-related side effects observed with antitoxins known presently.

When birds are used, it is contemplated that the antibody will be obtained from either the bird serum or the egg. A preferred embodiment involves collection of the antibody from the egg. Laying hens transport immunoglobulin to the egg yolk ("IgY") in concentrations equal to or exceeding that found in serum. See R. Patterson et al., J. Immunol. 89:272 (1962); and S. B. Carroll and B. D. Stollar, J. Biol. Chem. 258:24 (1983).! In addition, the large volume of egg yolk produced vastly exceeds the volume of serum that can be safely obtained from the bird over any given time period. Finally, the antibody from eggs is purer and more homogeneous; there is far less non-immunoglobulin protein (as compared to serum) and only one class of immunoglobulin is transported to the yolk.

When considering immunization with toxins, one may consider modification of the toxins to reduce the toxicity. In this regard, it is not intended that the present invention be limited by immunization with modified toxin. Unmodified ("native") toxin is also contemplated as an immunogen.

It is also not intended that the present invention be limited by the type of modification--if modification is used. The present invention contemplates all types of toxin modification, including chemical and heat treatment of the toxin. The preferred modification, however, is formaldehyde treatment.

It is not intended that the present invention be limited to a particular mode of immunization; the present invention contemplates all modes of immunization, including subcutaneous, intramuscular, intraperitoneal, and intravenous or intravascular injection, as well as per os administration of immunogen.

The present invention further contemplates immunization with or without adjuvant. (Adjuvant is defined as a substance known to increase the immune response to other antigens when administered with other antigens.) If adjuvant is used, it is not intended that the present invention be limited to any particular type of adjuvant--or that the same adjuvant, once used, be used all the time. While the present invention contemplates all types of adjuvant, whether used separately or in combinations, the preferred use of adjuvant is the use of Complete Freund's Adjuvant followed sometime later with Incomplete Freund's Adjuvant. Another preferred use of adjuvant is the use of Gerbu Adjuvant. The invention also contemplates the use of RIBI fowl adjuvant and Quil A adjuvant.

When immunization is used, the present invention contemplates a wide variety of immunization schedules. In one embodiment, a chicken is administered toxin(s) on day zero and subsequently receives toxin(s) in intervals thereafter. It is not intended that the present invention be limited by the particular intervals or doses. Similarly, it is not intended that the present invention be limited to any particular schedule for collecting antibody. The preferred collection time is sometime after day 100.

Where birds are used and collection of antibody is performed by collecting eggs, the eggs may be stored prior to processing for antibody. It is preferred that eggs be stored at 4° C. for less than one year.

It is contemplated that chicken antibody produced in this manner can be buffer-extracted and used analytically. While unpurified, this preparation can serve as a reference for activity of the antibody prior to further manipulations (e.g., immunoaffinity purification).

III. Increasing The Effectiveness Of Antibodies

When purification is used, the present invention contemplates purifying to increase the effectiveness of both non-mammalian antitoxins and mammalian antitoxins. Specifically, the present invention contemplates increasing the percent of toxin-reactive immunoglobulin. The preferred purification approach for avian antibody is polyethylene glycol (PEG) separation.

The present invention contemplates that avian antibody be initially purified using simple, inexpensive procedures. In one embodiment, chicken antibody from eggs is purified by extraction and precipitation with PEG. PEG purification exploits the differential solubility of lipids (which are abundant in egg yolks) and yolk proteins in high concentrations of PEG 8000. Polson et al., Immunol. Comm. 9:495 (1980).! The technique is rapid, simple, and relatively inexpensive and yields an immunoglobulin fraction that is significantly purer in terms of contaminating non-immunoglobulin proteins than the comparable ammonium sulfate fractions of mammalian sera and horse antibodies. The majority of the PEG is removed from the precipitated chicken immunoglobulin by treatment with ethanol. Indeed, PEG-purified antibody is sufficiently pure that the present invention contemplates the use of PEG-purified antitoxins in the passive immunization of intoxicated humans and animals.

The invention further contemplates increasing the effectiveness of compositions comprising antitoxins by enterically-coating a solid form of the antitoxin to improve the survival of the antitoxin in the gastrointestinal tract (i.e., enteric stability) as discussed further below in section IV(C).

IV. Treatment

The present invention contemplates antitoxin therapy for humans and other animals intoxicated by bacterial toxins. A preferred method of treatment is by oral administration of antitoxin. Another preferred method of treatment is by parenteral administration of antitoxin.

A. Therapeutic Preparations and Combinations

The present invention contemplates using therapeutic compositions of antitoxins. The antitoxin compositions may comprise antitoxin in a solid or liquid form.

It is not intended that the present invention be limited by the particular nature of the therapeutic preparation. For example, such compositions can be provided together with physiologically tolerable liquid, gel or solid carriers, diluents, adjuvants and excipients. In addition, the antitoxins may be used together with other therapeutic agents, including antibiotics.

As noted above, these therapeutic preparations can be administered to mammals for veterinary use, such as with domestic animals, and clinical use in humans in a manner similar to other therapeutic agents. In general, the dosage required for therapeutic efficacy will vary according to the type of use and mode of administration, as well as the particularized requirements of individual hosts.

With respect to the mode of administration, the antitoxins may be employed for oral, intravenous, intraperitoneal, intramuscular or intrathecal administration. Formulations for such administrations may comprise an effective amount of antitoxin in sterile water or physiological saline.

On the other hand, formulations may contain such normally employed additives such as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions typically contain 1%-95% of active ingredient, preferably 2%-70%.

The compositions are preferably prepared for oral administration, either as liquid solutions or suspensions; solid forms, including solid forms suitable for solution in, or suspension in, liquid prior to administration, may also be prepared. Solid forms of the antitoxins may further comprise an enteric coating. The compositions are also preferably prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to administration may also be prepared.

The antitoxins of the present invention are often mixed with diluents or excipients which are physiological tolerable and compatible. Suitable diluents and excipients are, for example, water, saline, nutritional formulations (e.g., Ensure®, Enfamil®, etc.) dextrose, glycerol, or the like, and combinations thereof. In addition, if desired the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH buffering agents.

B. Dosage Of Antitoxin

It is noted by way of background that a balance must be struck when administering currently available antitoxin which is usually produced in large animals such as horses; sufficient antitoxin must be administered to neutralize the toxin, but not so much antitoxin as to increase the risk of untoward side effects. These side effects are caused by: i) patient sensitivity to foreign (e.g, horse) proteins; ii) anaphylactic or immunogenic properties of non-immunoglobulin proteins; iii) the complement fixing properties of mammalian antibodies; and/or iv) the overall burden of foreign protein administered. It is extremely difficult to strike this balance when, as noted above, the degree of intoxication (and hence the level of antitoxin therapy needed) can only be approximated.

The present invention contemplates significantly reducing side effects so that this balance is more easily achieved. Treatment according to the present invention contemplates reducing side effects by using PEG-purified antitoxin from birds.

In one embodiment, the treatment of the present invention contemplates the use of PEG-purified antitoxin from birds. The use of yolk-derived, PEG-purified antibody as antitoxin allows for the administration of: 1) non(mammalian)-complement-fixing, avian antibody; 2) a less heterogeneous mixture of non-immunoglobulin proteins; and 3) less total protein to deliver the equivalent weight of active antibody present in currently available antitoxins. The non-mammalian source of the antitoxin makes it useful for treating patients who are sensitive to horse or other mammalian sera.

As is true in cases of botulism, the degree of an individual's exposure to C. difficile toxin and the prognosis are often difficult to assess, and depend upon a number of factors (e.g., the quantity of the inoculum, the toxigenicity and serotype of C. difficile strain involved, etc.). Thus, the clinical presentation of a patient is usually a more important consideration than a quantitative diagnostic test, for determination of dosage in antitoxin administration. Indeed, for many toxin-associated diseases (e.g., botulism, tetanus, diphtheria, etc.), there is no rapid, quantitative test to detect the presence of the toxin or organism. Rather, these toxin-associated diseases are medical emergencies which mandate immediate treatment. Confirmation of the etiologic agent must not delay the institution of therapy, as the condition of an affected patient may rapidly deteriorate. In addition to the initial treatment with antitoxin, subsequent doses may be indicated, as the patient's disease progresses. The dosage and timing of these subsequent doses is dependent upon the signs and symptoms of disease in each individual patient.

It is contemplated that when antitoxin is to be administered parentally, the administration of antitoxin to an affected individual would involve an initial injection of an approximately 10 ml dose of immune globulin (with less than approximately 1 gram of total protein). In one preferred embodiment, it is contemplated that at least 50% of the initial injection comprises immune globulin. It is also contemplated that more purified immune globulin be used for treatment, wherein approximately 90% of the initial injection comprises immune globulin. When more purified immune globulin (e.g., purified IgY) is used, it is contemplated that the total protein will be less than approximately 100 milligrams. It is also contemplated that additional doses be given, depending upon the signs and symptoms associated with C. difficile associated disease progression.

It is contemplated that when antitoxin is to be administered orally, the administration of antitoxin to an affected individual would involve a treatment course (i.e., initial and subsequent doses) comprising the administration of a therapeutic composition comprising about 50 gm of antitoxin and more preferably about 4-5 gm of antitoxin.

C. Delivery Of Antitoxin

Although it is not intended to limit the route of delivery, the present invention contemplates a method for antitoxin treatment of bacterial intoxication in which delivery of antitoxin is parenteral or oral.

In one embodiment, antitoxin is parenterally administered to a subject in an aqueous solution. It is not intended that the parenteral administration be limited to a particular route. Indeed, it is contemplated that all routes of parenteral administration will be used. In one embodiment, parenteral administration is accomplished via intramuscular injection. In an alternative embodiment, parenteral administration is accomplished via intravenous injection.

In one embodiment, antitoxin is delivered in a solid form (e.g., tablets, capsules). In an alternative embodiment antitoxin is delivered in an aqueous solution. When an aqueous solution is used, the solution has sufficient ionic strength to solubilize antibody protein, yet is made palatable for oral administration. The delivery solution may also be buffered (e.g., carbonate buffer pH 9.5) which can neutralize stomach acids and stabilize the antibodies when the antibodies are administered orally. In one embodiment the delivery solution is an aqueous solution. In another embodiment the delivery solution is a nutritional formula. Preferably, the delivery solution is infant formula. Yet another embodiment contemplates the delivery of lyophilized antibody encapsulated or microencapsulated inside acid-resistant compounds.

Methods of applying enteric coatings to pharmaceutical compounds are well known to the art companies specializing in the coating of pharmaceutical compounds are available; for example, The Coating Place (Verona, Wis.) and AAI (Wilmington, N.C.)!. Enteric coatings which are resistant to gastric fluid and whose release (i.e., dissolution of the coating to release the pharmaceutical compound) is pH dependent are commercially available for example, the polymethacrylates Eudragit® L and Eudragit® S (Rohm GmbH)!. Eudragit® S is soluble in intestinal fluid from pH 7.0; this coating can be used to microencapsulate lyophilized antitoxin antibodies and the particles are suspended in a solution having a pH above or below pH 7.0 for oral administration. The microparticles remain intact and undissolved until they reach the intestines where the intestinal pH causes them to dissolve thereby releasing the antitoxin.

The invention is directed to the improvement of the enteric stability of the therapeutic antitoxin Enteric stability is defined as the stability of the antitoxin during passage through the gastrointestinal tract; the enteric stability is improved by increasing the amount of the orally administered antitoxin which is delivered to the desired site (i.e., the intestines) in a functional or active form!. Antibodies, and avian antibodies in particular, are known to be significantly denatured when exposed to acidic solutions (e.g., gastric fluid). Denaturation of the antibody results in the loss of functionality (i.e., loss of the ability to bind to the specific target). In addition to the denaturation of antibodies due to the low pH found in portions of the gastrointestinal tract, proteolytic degradation of the antitoxin may occur due to digestion with enzymes. The invention improves the enteric stability of the therapeutic antitoxins by coating the antitoxins with an enteric coating. The enteric coating prevents the acid-induced denaturation of the antitoxin and prevents exposure of the antitoxin to enzymes present in the upper portions of the gastrointestinal tract.

Application of acid resistant enteric coatings are shown herein to prevent release of microencapsulated antitoxin (e.g., enterically-coated antitoxin) into simulated gastric solution while permitting release of the antitoxin in simulated intestinal solution. The enteric survival of the therapeutic antitoxins may also be improved through the use of excipients (more or less inert substances added to a therapeutic compound as a diluent or to give form or consistency when the compound is provided in tablet form). Excipients, such as carbonate buffers of about pH 9.5 or nutritional formulations (e.g., Ensure®, Enfamil®, etc.) may indirectly reduce the denaturation of the antitoxin in the stomach by raising the stomach pH or by providing additional protein to compete for degradation by gastric enzymes. In contrast, the use of enteric coatings on the antitoxin composition directly prevents the denaturation or digestion of the antitoxin in the stomach by preventing the release of the antitoxin from the enterically-coated particle until the particle reaches the intestinal fluid which has a basic pH. The use of enteric coatings is a particularly preferred means of improving the acid stability of the therapeutic antitoxins of the invention.

The invention contemplates a method of treatment which can be administered for treatment of acute intoxication. In one embodiment, antitoxin is administered orally in either a delivery solution or in tablet form, in therapeutic dosage, to a subject intoxicated by the bacterial toxin which served as immunogen for the antitoxin.

The invention also contemplates a method of treatment which can be administered prophylactically. In one embodiment, antitoxin is administered orally, in a delivery solution, in therapeutic dosage, to a subject, to prevent intoxication of the subject by the bacterial toxin which served as immunogen for the production of antitoxin. In another embodiment, antitoxin is administered orally in solid form such as tablets or as microencapsulated particles. Microencapsulation of lyophilized antibody using compounds such as Eudragit® (Rohm Tech, Inc.) or polyethylene glycol, which dissolve at a wide range of pH units, allows the oral administration of solid antitoxin in a liquid form (i.e., a suspension) to recipients unable to tolerate administration of tablets (e.g., children or patients on feeding tubes). In a preferred embodiment, the lyophilized antibody is coated with Eudragit® L30D (Rohm Tech, Inc.). In one preferred embodiment the subject is an child. In another embodiment, antibody raised against whole bacterial organism is administered orally to a subject, in a delivery solution, in therapeutic dosage.

V. Vaccines Against Clostridial Species

The invention contemplates the generation of mono- and multivalent vaccines for the protection of an animal (particularly humans) against several clostridial species. Of particular interest are vaccines which stimulate the production of a humoral immune response to C. difficile, C. tetani and C. botulinum in humans. The antigens comprising the vaccine preparation may be native or recombinantly produced toxin proteins from the clostridial species listed above. When toxin proteins are used as immunogens they are generally modified to reduce the toxicity. This modification may be by chemical or genetic (i.e., recombinant DNA technology) means. In general genetic detoxification (i.e., the expression of nontoxic fragments in a host cell) is preferred as the expression of nontoxic fragments in a host cell precludes the presence of intact, active toxin in the final preparation. However, when chemical modification is desired, the preferred toxin modification is formaldehyde treatment.

The invention contemplates that recombinant C. difficile toxin proteins be used as antigens in mono- and multivalent vaccine preparations. Soluble, substantially endotoxin-free recombinant C. difficile toxin A and or toxin B proteins may be used alone or in conjunction with either recombinant or native toxins or toxoids from C. botulinum, C. difficile and C. tetani as antigens for the preparation of these mono- and multivalent vaccines. It is contemplated that, due to the structural similarity of C. botulinum and C. tetani toxin proteins, a vaccine comprising C. difficile and botulinum toxin proteins (native or recombinant or a mixture thereof) be used to stimulate an immune response against C. botulinum, C. tetani and C. difficile.

The adverse consequences of exposure to C. difficile toxins would be avoided by immunization of subjects at risk of exposure to the toxin with nontoxic preparations which confer immunity such as chemically or genetically detoxified toxin.

Vaccines which confer immunity against one or more of the toxin types A and B would be useful as a means of protecting animals, including humans, from the deleterious effects of C. difficile toxins. A subject may be immunized with compositions comprising one or more C. difficile toxin proteins to generate neutralizing antibodies in the subject. A subject may be immunized with a first immunogen comprising C. difficile toxin A proteins followed by a separate immunization with a second immunogen comprising C. difficile B toxin proteins to produce neutralizing antibodies directed against C. difficile toxins A and B. Alternatively, the subject may be immunized with a single immunogen comprising C. difficile toxin A and B proteins.

In general, chemical detoxification of bacterial toxins using agents such as formaldehyde, glutaraldehyde or hydrogen peroxide is not optimal for the generation of vaccines or antitoxins. A delicate balance must be struck between too much and too little chemical modification. If the treatment is insufficient, the vaccine may retain residual toxicity. If the treatment is too excessive, the vaccine may lose potency due to destruction of native immunogenic determinants. Another major limitation of using botulinal toxoids for the generation of antitoxins or vaccines is the high production expense. For the above reasons, the development of methods for the production of nontoxic but immunogenic C. difficile toxin proteins is desirable.

Recombinant C. difficile toxin proteins have be produced in a host cell such as E. coli in either a soluble or insoluble form. Insoluble recombinant proteins are found in inclusion bodies. Inclusion body protein must be solubilized prior to purification and/or administration to a host. The harsh treatment of inclusion body protein needed to accomplish this solubilization may reduce the immunogenicity of the purified protein. Ideally, recombinant proteins to be used as vaccines are expressed as soluble proteins at high levels (i.e., greater than or equal to about 0.75% of total cellular protein) in E. coli or other host cells. This facilitates the production and isolation of sufficient quantities of the immunogen in a highly purified form (i.e., substantially free of endotoxin or other pyrogen contamination). The ability to express recombinant toxin proteins as soluble proteins in E. coli is advantageous due to the low cost of growth compared to insect or mammalian tissue culture cells.

The subject invention provides soluble C. difficile toxin proteins produced in economical host cells (e.g., E. coli). Further, methods for the isolation of purified soluble C. difficile toxin proteins which are suitable for immunization of humans and other animals are provided. These soluble, purified preparations of C. difficile toxin proteins provide the basis for improved vaccine preparations and facilitate the production of antitoxin.

When recombinant clostridial toxin proteins produced in gram-negative bacteria (e.g., E. coli) are used as vaccines, they are purified to remove endotoxin prior to administration to a host animal. In order to vaccinate a host, an immunogenically-effective amount of purified substantially endotoxin-free recombinant clostridial toxin protein is administered in any of a number of physiologically acceptable carriers known to the art. When administered for the purpose of vaccination, the purified substantially endotoxin-free recombinant clostridial toxin protein may be used alone or in conjunction with known adjutants, including potassium alum, aluminum phosphate, aluminum hydroxide, Gerbu adjuvant (GMDP; C.C. Biotech Corp.), RIBI adjuvant (MPL; RIBI Immunochemical Research, Inc.), QS21 (Cambridge Biotech). The alum and aluminum-based adjutants are particularly preferred when vaccines are to be administered to humans. The route of immunization may be nasal, oral, intramuscular, intraperitoneal or subcutaneous.

The invention contemplates the use of soluble, substantially endotoxin-free preparations of fusion proteins comprising portions of C. difficile toxins A and B as vaccines. In one embodiment, the vaccine comprises a portion of a C. difficile toxin and a poly-histidine tract (also called a histidine tag). In a particularly preferred embodiment, a fusion protein comprising a portion of a C. difficile toxin protein and a poly-histidine tract is expressed using the pET series of expression vectors (Novagen). The pET expression system utilizes a vector containing the T7 promoter which encodes the fusion protein and a host cell which can be induced to express the T7 DNA polymerase (i.e., a DE3 host strain). The production of C. difficile toxin fusion proteins containing a histidine tract is not limited to the use of a particular expression vector and host strain. Several commercially available expression vectors and host strains can be used to express the C. difficile protein sequences as a fusion protein containing a histidine tract (For example, the pQE series (pQE-8, 12, 16, 17, 18, 30, 31, 32, 40, 41, 42, 50, 51, 52, 60 and 70) of expression vectors (Qiagen) which are used with the host strains M15 pREP4! (Qiagen) and SG13009 pREP4! (Qiagen) can be used to express fusion proteins containing six histidine residues at the amino-terminus of the fusion protein).

VI. Detection Of Toxin

The invention contemplates detecting bacterial toxin in a sample. The term "sample" in the present specification and claims is used in its broadest sense. On the one hand it is meant to include a specimen or culture. On the other hand, it is meant to include both biological and environmental samples.

Biological samples may be animal, including human, fluid, solid (e.g., stool) or tissue; liquid and solid food products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Environmental samples include environmental material such as surface matter, soil, water and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, disposable and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention.

As discussed above in section IV, toxin-associated diseases are medical emergencies which mandate immediate treatment. Because existing methodologies do not provide rapid, quantitative tests for the presence of C. difficile toxins or organisms, treatment of subjects suspected of having C. difficile associated disease is begun prior to a determination of the amount or nature of the toxin or organism present. If a rapid and quantitative test for C. difficile toxins or organisms were available, the dosage of therapeutic compounds could be adjusted to provide maximum benefit to the intoxicated subject. The specific anti-C. difficile toxin A and B antibodies of the invention and the purified recombinant C. difficile toxin A and B proteins enable rapid and quantitative tests for C. difficile toxins or organisms.

The invention contemplates detecting bacterial toxin by a competitive immunoassay method that utilizes recombinant toxin A and toxin B proteins, antibodies raised against recombinant bacterial toxin proteins. A fixed amount of the recombinant toxin proteins are immobilized to a solid support (e.g., a microtiter plate) followed by the addition of a biological sample suspected of containing a bacterial toxin. The biological sample is first mixed with affinity-purified or PEG fractionated antibodies directed against the recombinant toxin protein. A reporter reagent is then added which is capable of detecting the presence of antibody bound to the immobilized toxin protein. The reporter substance may comprise an antibody with binding specificity for the antitoxin attached to a molecule which is used to identify the presence of the reporter substance. If toxin is present in the sample, this toxin will compete with the immobilized recombinant toxin protein for binding to the anti-recombinant antibody thereby reducing the signal obtained following the addition of the reporter reagent. A control is employed where the antibody is not mixed with the sample. This gives the highest (or reference) signal.

The invention also contemplates detecting bacterial toxin by a "sandwich" immunoassay method that utilizes antibodies directed against recombinant bacterial toxin proteins. Affinity-purified antibodies directed against recombinant bacterial toxin proteins are immobilized to a solid support (e.g., microtiter plates). Biological samples suspected of containing bacterial toxins are then added followed by a washing step to remove substantially all unbound antitoxin. The biological sample is next exposed to the reporter substance, which binds to antitoxin and is then washed free of substantially all unbound reporter substance. The reporter substance may comprise an antibody with binding specificity for the antitoxin attached to a molecule which is used to identify the presence of the reporter substance. Identification of the reporter substance in the biological tissue indicates the presence of the bacterial toxin.

It is also contemplated that bacterial toxin be detected by pouring liquids (e.g., soups and other fluid foods and feeds including nutritional supplements for humans and other animals) over immobilized antibody which is directed against the bacterial toxin. It is contemplated that the immobilized antibody will be present in or on such supports as cartridges, columns, beads, or any other solid support medium. In one embodiment, following the exposure of the liquid to the immobilized antibody, unbound toxin is substantially removed by washing. The exposure of the liquid is then exposed to a reporter substance which detects the presence of bound toxin. In a preferred embodiment the reporter substance is an enzyme, fluorescent dye, or radioactive compound attached to an antibody which is directed against the toxin (i.e., in a "sandwich" immunoassay). It is also contemplated that the detection system will be developed as necessary (e.g., the addition of enzyme substrate in enzyme systems; observation using fluorescent light for fluorescent dye systems; and quantitation of radioactivity for radioactive systems).

Experimental

The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

In the disclosure which follows, the following abbreviations apply: °C. (degrees Centigrade); rpm (revolutions per minute); BBS-Tween (borate buffered saline containing Tween); BSA (bovine serum albumin); ELISA (enzyme-linked immunosorbent assay); CFA (complete Freund's adjuvant); IFA (incomplete Freund's adjuvant); IgG (immunoglobulin G); IgY (immunoglobulin Y); IM (intramuscular); IP (intraperitoneal); IV (intravenous or intravascular); SC (subcutaneous); H₂ O (water); HCl (hydrochloric acid); LD₁₀₀ (lethal dose for 100% of experimental animals); aa (amino acid); HPLC (high performance liquid chromatography); kD (kilodaltons); gm (grams); μg (micrograms); mg (milligrams); ng (nanograms); μl (microliters); ml (milliliters); mm (millimeters); nm (nanometers); μm (micrometer); M (molar); mM (millimolar); MW (molecular weight); sec (seconds); min(s) (minute/minutes); hr(s) (hour/hours); MgCl₂ (magnesium chloride); NaCl (sodium chloride); Na₂ CO₃ (sodium carbonate); OD₂₈₀ (optical density at 280 nm); OD₆₀₀ (optical density at 600 nm); PAGE (polyacrylamide gel electrophoresis); PBS phosphate buffered saline (150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2)!; PEG (polyethylene glycol); PMSF (phenylmethylsulfonyl fluoride); SDS (sodium dodecyl sulfate); Tris (tris(hydroxymethyl)aminomethane); Ensure® (Ensure®, Ross Laboratories, Columbus Ohio); Enfamil® (Enfamil®, Mead Johnson); w/v (weight to volume); v/v (volume to volume); Accurate Chemical (Accurate Chemical & Scientific Corp., Westbury, N.Y.); Amicon (Amicon, Inc., Beverly, Mass.); Amresco (Amresco, Inc., Solon, Ohio); ATCC (American Type Culture Collection, Rockville, Md.); BBL (Baltimore Biologics Laboratory, (a division of Becton Dickinson), Cockeysville, Md.); Becton Dickinson (Becton Dickinson Labware, Lincoln Park, N.J.); BioRad (BioRad, Richmond, Calif.); Biotech (C--C Biotech Corp., Poway, Calif.); Charles River (Charles River Laboratories, Wilmington, Mass.); Cocalico (Cocalico Biologicals Inc., Reamstown, Pa.); CytRx (CytRx Corp., Norcross, Ga.); Falcon (e.g. Baxter Healthcare Corp., McCaw Park, Ill. and Becton Dickinson); FDA (Federal Food and Drug Administration); Fisher Biotech (Fisher Biotech, Springfield, N.J.); GIBCO (Grand Island Biologic Company/BRL, Grand Island, N.Y.); Gibco-BRL (Life Technologies, Inc., Gaithersburg, Md.); Harlan Sprague Dawley (Harlan Sprague Dawley, Inc., Madison, Wis.); Mallinckrodt (a division of Baxter Healthcare Corp., McGaw Park, Ill.); Millipore (Millipore Corp., Marlborough, Mass.); New England Biolabs (New England Biolabs, Inc., Beverly, Mass.); Novagen (Novagen, Inc., Madison, Wis.); Pharmacia (Pharmacia, Inc., Piscataway, N.J.); Qiagen (Qiagen, Chatsworth, Calif.); RIBI (RIBI Immunochemical Research, Inc., Hamilton, Mont.); Sasco (Sasco, Omaha, Nebr.); Showdex (Showa Denko America, Inc., New York, N.Y.); Sigma (Sigma Chemical Co., St. Louis, Mo.); Sterogene (Sterogene, Inc., Arcadia, Calif.); Tech Lab (Tech Lab, Inc., Blacksburg, Va.); and Vaxcell (Vaxcell, Inc., a subsidiary of CytRX Corp., Norcross, Calif.).

When a recombinant protein is described in the specification it is referred to in a short-hand manner by the amino acids in the toxin sequence present in the recombinant protein rounded to the nearest 10. For example, the recombinant protein pMB1850-2360 contains amino acids 1852 through 2362 of the C. difficile toxin B protein. The specification gives detailed construction details for all recombinant proteins such that one skilled in the art will know precisely which amino acids are present in a given recombinant protein.

EXAMPLE 1 Production Of High-Titer Antibodies To Clostridium difficile Organisms In A Hen

Antibodies to certain pathogenic organisms have been shown to be effective in treating diseases caused by those organisms. It has not been shown whether antibodies can be raised, against Clostridium difficile, which would be effective in treating infection by this organism. Accordingly, C. difficile was tested as immunogen for production of hen antibodies.

To determine the best course for raising high-titer egg antibodies against whole C. difficile organisms, different immunizing strains and different immunizing concentrations were examined. The example involved (a) preparation of the bacterial immunogen, (b) immunization, (c) purification of anti-bacterial chicken antibodies, and (d) detection of anti-bacterial antibodies in the purified IgY preparations.

a) Preparation Of Bacterial Immunogen

C. difficile strains 43594 (serogroup A) and 43596 (serogroup C) were originally obtained from the ATCC. These two strains were selected because they represent two of the most commonly-occurring serogroups isolated from patients with antibiotic-associated pseudomembranous colitis. Delmee et al., J. Clin. Microbiol., 28(10):2210 (1990).! Additionally, both of these strains have been previously characterized with respect to their virulence in the Syrian hamster model for C. difficile infection. Delmee et al., J. Med Microbiol., 33:85 (1990).!

The bacterial strains were separately cultured on brain heart infusion agar for 48 hours at 37° C. in a Gas Pack 100 Jar (BBL, Cockeysville, Md.) equipped with a Gas Pack Plus anaerobic envelope (BBL). Forty-eight hour cultures were used because they produce better growth and the organisms have been found to be more cross-reactive with respect to their surface antigen presentation. The greater the degree of cross-reactivity of our IgY preparations, the better the probability of a broad range of activity against different strains/serogroups. Toma et al., J. Clin. Microbiol., 26(3):426 (1988).!

The resulting organisms were removed from the agar surface using a sterile dacron-tip swab, and were suspended in a solution containing 0.4% formaldehyde in PBS, pH 7.2. This concentration of formaldehyde has been reported as producing good results for the purpose of preparing whole-organism immunogen suspensions for the generation of polyclonal anti-C. difficile antisera in rabbits. Delmee et al., J. Clin. Microbiol., 21:323 (1985); Davies et al., Microbial Path., 9:141 (1990).! In this manner, two separate bacterial suspensions were prepared, one for each strain. The two suspensions were then incubated at 4° C. for 1 hour. Following this period of formalin-treatment, the suspensions were centrifuged at 4,200×g for 20 min., and the resulting pellets were washed twice in normal saline. The washed pellets, which contained formalin-treated whole organisms, were resuspended in fresh normal saline such that the visual turbidity of each suspension corresponded to a #7 McFarland standard. M. A. C. Edelstein, "Processing Clinical Specimens for Anaerobic Bacteria: Isolation and Identification Procedures," in S. M. Finegold et al (eds.)., Bailey and Scott's Diagnostic Microbiology, pp. 477-507, C. V. Mosby Co., (1990). The preparation of McFarland nephelometer standards and the corresponding approximate number of organisms for each tube are described in detail at pp. 172-173 of this volume.! Each of the two #7 suspensions was then split into two separate volumes. One volume of each suspension was volumetrically adjusted, by the addition of saline, to correspond to the visual turbidity of a #1 McFarland standard. Id.! The #1 suspensions contained approximately 3×10⁸ organisms/ml, and the #7 suspensions contained approximately 2×10⁹ organisms/mi. Id.! The four resulting concentration-adjusted suspensions of formalin-treated C. difficile organisms were considered to be "bacterial immunogen suspensions." These suspensions were used immediately after preparation for the initial immunization. See section (b).!

The formalin-treatment procedure did not result in 100% non-viable bacteria in the immunogen suspensions. In order to increase the level of killing, the formalin concentration and length of treatment were both increased for subsequent immunogen preparations, as described below in Table 3. (Although viability was decreased with the stronger formalin treatment, 100% inviability of the bacterial immunogen suspensions was not reached.) Also, in subsequent immunogen preparations, the formalin solutions were prepared in normal saline instead of PBS. At day 49, the day of the fifth immunization, the excess volumes of the four previous bacterial immunogen suspensions were stored frozen at -70° C. for use during all subsequent immunizations.

b) Immunization

For the initial immunization, 1.0 ml volumes of each of the four bacterial immunogen suspensions described above were separately emulsified in 1.2 ml volumes of CFA (GIBCO). For each of the four emulsified immunogen suspensions, two four-month old White Leghorn hens (pre-laying) were immunized. (It is not necessary to use pre-laying hens; actively-laying hens can also be utilized.) Each hen received a total volume of approximately 1.0 ml of a single emulsified immunogen suspension via four injections (two subcutaneous and two intramuscular) of approximately 250 μl per site. In this manner, a total of four different immunization combinations, using two hens per combination, were initiated for the purpose of evaluating both the effect of immunizing concentration on egg yolk antibody (IgY) production, and interstrain cross-reactivity of IgY raised against heterologous strains. The four immunization groups are summarized in Table 3.

The time point for the first series of immunizations was designated as "day zero." All subsequent immunizations were performed as described above except that the bacterial immunogen suspensions were emulsified using IFA (GIBCO) instead of

                  TABLE 3                                                          ______________________________________                                         Immunization Groups                                                            GROUP        IMMUNIZING APPROXIMATE                                            DESIGNATION  STRAIN     IMMUNIZING DOSE                                        ______________________________________                                         CD 43594, #1 C. difficile                                                                              1.5 × 10.sup.8 organisms/hen                                  strain 43594                                                      CD 43594, #7 C. difficile                                                                              1.0 × 10.sup.9 organisms/hen                                  strain 43594                                                      CD 43596, #1 C. difficile                                                                              1.5 × 10.sup.8 organisms/hen                                  strain 43596                                                      CD 43596, #7 C. difficile                                                                              1.0 × 10.sup.9 organisms/hen                                  strain 43596                                                      ______________________________________                                    

CFA, and for the later time point immunization, the stored frozen suspensions were used instead of freshly-prepared suspensions. The immunization schedule used is listed in Table 4.

c) Purification Of Anti-Bacterial Chicken Antibodies

Groups of four eggs were collected per immunization group between days 80 and 84 post-initial immunization, and chicken immunoglobulin (IgY) was extracted according to a modification of the procedure of A. Polson et al., Immunol. Comm., 9:495 (1980). A gentle stream of distilled water from a squirt bottle was used to separate the yolks from the whites, and the yolks were broken by dropping them through a funnel into a graduated cylinder. The four individual yolks were pooled for each group. The pooled, broken yolks were blended with 4 volumes of egg extraction buffer to improve antibody yield (egg extraction buffer is 0.01M sodium phosphate, 0.1M NaCl, pH 7.5, containing 0.005% thimerosal), and PEG 8000 (Amresco) was

                  TABLE 4                                                          ______________________________________                                         Immunization Schedule                                                          DAY OF       FORMALIN-  IMMUNOGEN                                              IMMUNIZATION TREATMENT  PREPARATION USED                                       ______________________________________                                         0            1%, 1 hr.  freshly-prepared                                       14           1%, overnight                                                                             "                                                      21           1%, overnight                                                                             "                                                      35           1%, 48 hrs.                                                                               "                                                      49           1%, 72 hrs.                                                                               "                                                      70           1%, 72 hrs.                                                                               stored frozen                                          85           1%, 72 hrs.                                                                               "                                                      105          1%, 72 hrs.                                                                               "                                                      ______________________________________                                    

added to a concentration of 3.5%. When all the PEG dissolved, the protein precipitates that formed were pelleted by centrifugation at 13,000×g for 10 minutes. The supernatants were decanted and filtered through cheesecloth to remove the lipid layer, and the PEG was added to the supernatants to a final concentration of 12% (the supernatants were assumed to contain 3.5% PEG). After a second centrifugation, the supernatants were discarded and the pellets were centrifuged a final time to extrude the remaining PEG. These crude IgY pellets were then dissolved in the original yolk volume of egg extraction buffer and stored at 4° C. As an additional control, a preimmune IgY solution was prepared as described above, using eggs collected from unimmunized hens.

d) Detection Of Anti-Bacterial Antibodies In The Purified IgY Preparations

In order to evaluate the relative levels of specific anti-C. difficile activity in the IgY preparations described above, a modified version of the whole-organism ELISA procedure of N. V. Padhye et al., J. Clin. Microbiol. 29:99-103 (1990) was used. Frozen organisms of both C. difficile strains described above were thawed and diluted to a concentration of approximately 1×10⁷ organisms/ml using PBS, pH 7.2. In this way, two separate coating suspensions were prepared, one for each immunizing strain. Into the wells of 96-well microtiter plates (Falcon, Pro-Bind Assay Plates) were placed 100 μl volumes of the coating suspensions. In this manner, each plate well received a total of approximately 1×10⁶ organisms of one strain or the other. The plates were then incubated at 4° C. overnight. The next morning, the coating suspensions were decanted, and all wells were washed three times using PBS. In order to block non-specific binding sites, 100 μl of 0.5% BSA (Sigma) in PBS was then added to each well, and the plates were incubated for 2 hours at room temperature. The blocking solution was decanted, and 100 μl volumes of the IgY preparations described above were initially diluted 1:500 with a solution of 0.1% BSA in PBS, and then serially diluted in 1:5 steps. The following dilutions were placed in the wells: 1:500, 1:2,500, 1:62,5000, 1:312,500, and 1:1,562,500. The plates were again incubated for 2 hours at room temperature. Following this incubation, the IgY-containing solutions were decanted, and the wells were washed three times using BBS-Tween (0.1M boric acid, 0.025M sodium borate, 1.0M NaCl, 0.1% Tween-20), followed by two washes using PBS-Tween (0.1% Tween-20), and finally, two washes using PBS only. To each well, 100 μl of a 1:750 dilution of rabbit anti-chicken IgG (whole-molecule)-alkaline phosphatase conjugate (Sigma) (diluted in 0.1% BSA in PBS) was added. The plates were again incubated for 2 hours at room temperature. The conjugate solutions were decanted and the plates were washed as described above, substituting 50 mM Na₂ CO₃, pH 9.5 for the PBS in the final wash. The plates were developed by the addition of 100 μl of a solution containing 1 mg/ml para-nitrophenyl phosphate (Sigma) dissolved in 50 mM Na₂ CO₃, 10 mM MgCl₂, pH 9.5 to each well, and incubating the plates at

                  TABLE 5                                                          ______________________________________                                         Results Of The Anti-C. difficile Whole-Organism ELISA                          IgY       DILUTION OF                                                                               43594-COATED                                                                               43596-COATED                                  PREPARATION                                                                              IgY PREP   WELLS       WELLS                                         ______________________________________                                         CD 43594, #1                                                                             1:500      1.746       1.801                                                   1:2,500    l.092       1.670                                                   1:12,500   0.202       0.812                                                   1:62,500   0.136       0.179                                                   1:312,500  0.012       0.080                                                   1:1,562,500                                                                               0.002       0.020                                         CD 43594, #7                                                                             1:500      1.780       1.771                                                   1:2,500    1.025       1.078                                                   1:12,500   0.188       0.382                                                   1:62,500   0.052       0.132                                                   1:312,500  0.022       0.043                                                   1:1,562,500                                                                               0.005       0.024                                         CD 43596, #1                                                                             1:500      1.526       1.790                                                   1:2,500    0.832       1.477                                                   1:12,500   0.247       0.452                                                   1:62,500   0.050       0.242                                                   1:312,500  0.010       0.067                                                   1:1,562,500                                                                               0.000       0.036                                         CD 43596, #7                                                                             1:500      1.702       1.505                                                   1:2,500    0.706       0.866                                                   1:12,500   0.250       0.282                                                   1:62,500   0.039       0.078                                                   1:312,500  0.002       0.017                                                   1:1,562,500                                                                               0.000       0.010                                         Preimmune IgY                                                                            1:500      0.142       0.309                                                   1:2,500    0.032       0.077                                                   1:12,500   0.006       0.024                                                   1:62,500   0.002       0.012                                                   1:312,500  0.004       0.010                                                   1:1,562,500                                                                               0.002       0.014                                         ______________________________________                                    

room temperature in the dark for 45 minutes. The absorbance of each well was measured at 410 mn using a Dynatech MR 700 plate reader. In this manner, each of the four IgY preparations described above was tested for reactivity against both of the immunizing C. difficile strains; strain-specific, as well as cross-reactive activity was determined.

Table 5 shows the results of the whole-organism ELISA. All four IgY preparations demonstrated significant levels of activity, to a dilution of 1:62,500 or greater against both of the immunizing organism strains. Therefore, antibodies raised against one strain were highly cross-reactive with the other strain, and vice versa. The immunizing concentration of organisms did not have a significant effect on organism-specific IgY production, as both concentrations produced approximately equivalent responses. Therefore, the lower immunizing concentration of approximately 1.5×10⁸ organisms/hen is the preferred immunizing concentration of the two tested. The preimmune IgY preparation appeared to possess relatively low levels of C. difficile-reactive activity to a dilution of 1:500, probably due to prior exposure of the animals to environmental clostridia.

An initial whole-organism ELISA was performed using IgY preparations made from single CD 43594, #1 and CD 43596, #1 eggs collected around day 50 (data not shown). Specific titers were found to be 5 to 10-fold lower than those reported in Table 5. These results demonstrate that it is possible to begin immunizing hens prior to the time that they begin to lay eggs, and to obtain high titer specific IgY from the first eggs that are laid. In other words, it is not necessary to wait for the hens to begin laying before the immunization schedule is started.

EXAMPLE 2 Treatment Of C. difficile Infection With Anti-C. difficile Antibody

In order to determine whether the immune IgY antibodies raised against whole C. difficile organisms were capable of inhibiting the infection of hamsters by C. difficile, hamsters infected by these bacteria were utilized. Lyerly et al., Infect. Immun., 59:2215-2218 (1991).! This example involved: (a) determination of the lethal dose of C. difficile organisms; and (b) treatment of infected animals with immune antibody or control antibody in nutritional solution.

a) Determination Of The Lethal Dose Of C. difficile Organisms

Determination of the lethal dose of C. difficile organisms was carried out according to the model described by D. M. Lyerly et al., Infect. Immun., 59:2215-2218 (1991). C. difficile strain ATCC 43596 (serogroup C, ATCC) was plated on BHI agar and grown anaerobically (BBL Gas Pak 100 system) at 37° C. for 42 hours. Organisms were removed from the agar surface using a sterile dacron-tip swab and suspended in sterile 0.9% NaCl solution to a density of 10⁸ organisms/ml.

In order to determine the lethal dose of C. difficile in the presence of control antibody and nutritional formula, non-immune eggs were obtained from unimmunized hens and a 12% PEG preparation made as described in Example 1(c). This preparation was redissolved in one fourth the original yolk volume of vanilla flavor Ensure®.

Starting on day one, groups of female Golden Syrian hamsters (Harlan Sprague Dawley), 8-9 weeks old and weighing approximately 100 gm, were orally administered 1 ml of the preimmune/Ensure® formula at time zero, 2 hours, 6 hours, mad 10 hours. At 1 hour, animals were orally administered 3.0 mg clindamycin HCl (Sigma) in 1 ml of water. This drug predisposes hamsters to C. difficile infection by altering the normal intestinal flora. On day two, the animals were given 1 ml of the preimmune IgY/Ensure® formula at time zero, 2 hours, 6 hours, and 10 hours. At 1 hour on day two, different groups of animals were inoculated orally with saline (control), or 10², 10⁴, 10⁶, or 10⁸ C. difficile organisms in 1 ml of saline. From days 3-12, animals were given 1 ml of the preimmune IgY/Ensure® formula three times daily and observed for the onset of diarrhea and death. Each animal was housed in an individual cage and was offered food and water ad libitum.

Administration of 10⁶ -10⁸ organisms resulted in death in 3-4 days while the lower doses of 10² -10⁴ organisms caused death in 5 days. Cecal swabs taken from dead animals indicated the presence of C. difficile. Given the effectiveness of the 10² dose, this number of organisms was chosen for the following experiment to see if hyperimmune anti-C. difficile antibody could block infection.

b) Treatment Of Infected Animals With Immune Antibody Or Control Antibody In Nutritional Formula

The experiment in (a) was repeated using three groups of seven hamsters each. Group A received no clindamycin or C. difficile and was the survival control. Group B received clindamycin, 10² C. difficile organisms and preimmune IgY on the same schedule as the animals in (a) above. Group C received clindamycin, 10² C. difficile organisms, and hyperimmune anti-C. difficile IgY on the same schedule as Group B. The anti-C. difficile IgY was prepared as described in Example 1 except that the 12% PEG preparation was dissolved in one fourth the original yolk volume of Ensure®.

All animals were observed for the onset of diarrhea or other disease symptoms and death. Each animal was housed in an individual cage and was offered food and water ad libitum. The results are shown in Table 6.

Hamsters in the control group A did not develop diarrhea and remained healthy during the experimental period. Hamsters in groups B and C developed diarrheal disease. Anti-C. difficile IgY did not protect the animals from diarrhea or death, all animals succumbed in the same time interval as the animals treated with preimmune

                  TABLE 6                                                          ______________________________________                                         The Effect Of Oral Feeding Of Hyperimmune IgY Antibody                         on C. difficile Infection                                                                         TIME TO    TIME TO                                          ANIMAL GROUP       DIARRHEA.sup.a                                                                            DEATH.sup.a                                      ______________________________________                                         A      pre-immune IgY only                                                                            no diarrhea                                                                               no deaths                                    B      Clindamycin, C. difficile,                                                                     30 hrs.    49 hrs.                                             preimmune IgY                                                           C      Clindamycin, C. difficile,                                                                     33 hrs.    56 hrs.                                             immune IgY                                                              ______________________________________                                          .sup.a Mean of seven animals.                                            

IgY. Thus, while immunization with whole organisms apparently can improve sub-lethal symptoms with particular bacteria (see U.S. Pat. No. 5,080,895 to H. Tokoro), such an approach does not prove to be productive to protect against the lethal effects of C. difficile.

EXAMPLE 3 Production of C. botulinum Type A Antitoxin in Hens

In order to determine whether antibodies could be raised against the toxin produced by clostridial pathogens, which would be effective in treating clostridial diseases, antitoxin to C. botulinum type A toxin was produced. This example involves: (a) toxin modification; (b) immunization; (c) antitoxin collection; (d) antigenicity assessment; and (e) assay of antitoxin titer.

a) Toxin Modification

C. botulinum type A toxoid was obtained from B. R. DasGupta. From this, the active type A neurotoxin (M.W. approximately 150 kD) was purified to greater than 99% purity, according to published methods. B. R. DasGupta & V. Sathyamoorthy, Toxicon, 22:415 (1984).! The neurotoxin was detoxified with formaldehyde according to published methods. B. R. Singh & B. R. DasGupta, Toxicon, 27:403 (1989).!

b) Immunization

C. botulinum toxoid for immunization was dissolved in PBS (1 mg/ml) and was emulsified with an approximately equal volume of CFA (GIBCO) for initial immunization or IFA for booster immunization. On day zero, two white leghorn hens, obtained from local breeders, were each injected at multiple sites (intramuscular and subcutaneous) with 1 ml inactivated toxoid emulsified in 1 ml CFA. Subsequent booster immunizations were made according to the following schedule for day of injection and toxoid amount: days 14 and 21--0.5 mg; day 171--0.75 mg; days 394, 401, 409--0.25 mg. One hen received an additional booster of 0.150 mg on day 544.

c) Antitoxin Collection

Total yolk immunoglobulin (IgY) was extracted as described in Example 1(c) and the IgY pellet was dissolved in the original yolk volume of PBS with thimerosal.

d) Antigenicity Assessment

Eggs were collected from day 409 through day 423 to assess whether the toxoid was sufficiently immunogenic to raise antibody. Eggs from the two hens were pooled and antibody was collected as described in the standard PEG protocol. Example 1(c).! Antigenicity of the botulinal toxin was assessed on Western blots. The 150 kD detoxified type A neurotoxin and unmodified, toxic, 300 kD botulinal type A complex (toxin used for intragastric route administration for animal gut neutralization experiments; see Example 6) were separated on a SDS-polyacrylamide reducing gel. The Western blot technique was performed according to the method of Towbin. H. Towbin et at., Proc. Natl. Acad. Sci. USA, 76:4350 (1979).! Ten μg samples of C. botulinum complex and toxoid were dissolved in SDS reducing sample buffer (1% SDS, 0.5% 2-mercaptoethanol, 50 mM Tris, pH 6.8, 10% glycerol, 0.025% w/v bromphenol blue, 10% β-mercaptoethanol), heated at 95° C. for 10 min and separated on a 1 mm thick 5% SDS-polyacrylamide gel. K. Weber and M. Osborn, "Proteins and Sodium Dodecyl Sulfate: Molecular Weight Determination on Polyacrylamide Gels and Related Procedures," in The Proteins, 3d Edition (H. Neurath & R. L. Hill, eds), pp. 179-223, (Academic Press, NY, 1975).! Part of the gel was cut off and the proteins were stained with Coomassie Blue. The proteins in the remainder of the gel were transferred to nitrocellulose using the Milliblot-SDE electro-blotting system (Millipore) according to manufacturer's directions. The nitrocellulose was temporarily stained with 10% Ponceau S S. B. Carroll and A. Laughon, "Production and Purification of Polyclonal Antibodies to the Foreign Segment of β-galactosidase Fusion Proteins," in DNA Cloning: A Practical Approach, Vol. III, (D. Glover, ed.), pp. 89-111, IRL Press, Oxford, (1987)! to visualize the lanes, then destained by running a gentle stream of distilled water over the blot for several minutes. The nitrocellulose was immersed in PBS containing 3% BSA overnight at 4° C. to block any remaining protein binding sites.

The blot was cut into strips and each strip was incubated with the appropriate primary antibody. The avian anti-C. botulinum antibodies described in (c)! and preimmune chicken antibody (as control) were diluted 1:125 in PBS containing 1 mg/ml BSA for 2 hours at room temperature. The blots were washed with two changes each of large volumes of PBS, BBS-Tween and PBS, successively (10 min/wash). Goat anti-chicken IgG alkaline phosphatase conjugated secondary antibody (Fisher Biotech) was diluted 1:500 in PBS containing 1 mg/ml BSA and incubated with the blot for 2 hours at room temperature. The blots were washed with two changes each of large volumes of PBS and BBS-Tween, followed by one change of PBS and 0.1M Tris-HCl, pH 9.5. Blots were developed in freshly prepared alkaline phosphatase substrate buffer (100 μg/ml nitroblue tetrazolium (Sigma), 50 μg/ml 5-bromo-4-chloro-3-indolyl phosphate (Sigma), 5 mM MgCl₂ in 50 mM Na₂ CO₃, pH 9.5).

The Western blots are shown in FIG. 1. The anti-C. botulinum IgY reacted to the toxoid to give a broad immunoreactive band at about 145-150 kD on the reducing gel. This toxoid is refractive to disulfide cleavage by reducing agents due to formalin crosslinking. The immune IgY reacted with the active toxin complex, a 97 kD C. botulinum type A heavy chain and a 53 kD light chain. The preimmune IgY was unreactive to the C. botulinum complex or toxoid in the Western blot.

e) Antitoxin Antibody Titer

The IgY antibody titer to C. botulinum type A toxoid of eggs harvested between day 409 and 423 was evaluated by ELISA, prepared as follows. Ninety-six-well Falcon Pro-bind plates were coated overnight at 4° C. with 100 μl/well toxoid B. R. Singh & B. R. Das Gupta, Toxicon 27:403 (1989)! at 2.5 μg/ml in PBS, pH 7.5 containing 0.005% thimerosal. The following day the wells were blocked with PBS containing 1% BSA for 1 hour at 37° C. The IgY from immune or preimmune eggs was diluted in PBS containing 1% BSA and 0.05% Tween 20 mad the plates were incubated for 1 hour at 37° C. The plates were washed three times with PBS containing 0.05% Tween 20 and three times with PBS alone. Alkaline phosphatase-conjugated goat-anti-chicken IgG (Fisher Biotech) was diluted 1:750 in PBS containing 1% BSA and 0.05% Tween 20, added to the plates, and incubated 1 hour at 37° C. The plates were washed as before, and p-nitrophenyl phosphate (Sigma) at 1 mg/ml in 0.05M Na₂ CO₃, pH 9.5, 10 mM MgCl₂ was added.

The results are shown in FIG. 2. Chickens immunized with the toxoid generated high titers of antibody to the immunogen. Importantly, eggs from both immunized hens had significant anti-immunogen antibody titers as compared to preimmune control eggs. The anti-C. botulinum IgY possessed significant activity, to a dilution of 1:93,750 or greater.

EXAMPLE 4 Preparation Of Avian Egg Yolk Immunoglobulin In An Orally Administrable Form

In order to administer avian IgY antibodies orally to experimental mice, an effective delivery formula for the IgY had to be determined. The concern was that if the crude IgY was dissolved in PBS, the saline in PBS would dehydrate the mice, which might prove harmful over the duration of the study. Therefore, alternative methods of oral administration of IgY were tested. The example involved: (a) isola-tion of immune IgY; (b) solubilization of IgY in water or PBS, including subsequent dialysis of the IgY-PBS solution with water to eliminate or reduce the salts (salt and phosphate) in the buffer; and (c) comparison of the quantity and activity of recovered IgY by absorbance at 280 nm and PAGE, and enzyme-linked immunoassay (ELISA).

a) Isolation Of Immune IgY

In order to investigate the most effective delivery formula for IgY, we used IgY which was raised against Crotalus durissus terrificus venom. Three eggs were collected from hens immunized with the C. durissus terrificus venom and IgY was extracted from the yolks using the modified Polson procedure described by Thalley and Carroll Bio/Technology, 8:934-938 (1990)! as described in Example 1(c).

The egg yolks were separated from the whites, pooled, and blended with four volumes of PBS. Powdered PEG 8000 was added to a concentration of 3.5%. The mixture was centrifuged at 10,000 rpm for 10 minutes to pellet the precipitated protein, and the supernatant was filtered through cheesecloth to remove the lipid layer. Powdered PEG 8000 was added to the supernatant to bring the final PEG concentration to 12% (assuming a PEG concentration of 3.5% in the supernatant). The 12% PEG/IgY mixture was divided into two equal volumes and centrifuged to pellet the IgY.

b) Solubilization Of The IgY In Water Or PBS

One pellet was resuspended in 1/2 the original yolk volume of PBS, and the other pellet was resuspended in 1/2 the original yolk volume of water. The pellets were then centrifuged to remove any particles or insoluble material. The IgY in PBS solution dissolved readily but the fraction resuspended in water remained cloudy.

In order to satisfy anticipated sterility requirements for orally administered antibodies, the antibody solution needs to be filter-sterilized (as an alternative to heat sterilization which would destroy the antibodies). The preparation of IgY resuspended in water was too cloudy to pass through either a 0.2 or 0.45 μm membrane filter, so 10 ml of the PBS resuspended fraction was dialyzed overnight at room temperature against 250 ml of water. The following morning the dialysis chamber was emptied and refilled with 250 ml of fresh H₂ O for a second dialysis. Thereafter, the yields of soluble antibody were determined at OD₂₈₀ and are compared in Table 7.

                  TABLE 7                                                          ______________________________________                                         Dependence Of IgY Yield On Solvents                                                           ABSORBANCE OF 1:10                                                                             PERCENT                                         FRACTION       DILUTION AT 280 nm                                                                             RECOVERY                                        ______________________________________                                         PBS dissolved  1.149           100%                                            H.sub.2 O dissolved                                                                           0.706           61%                                             PBS dissolved/H.sub.2 O dialyzed                                                              0.885           77%                                             ______________________________________                                    

Resuspending the pellets in PBS followed by dialysis against water recovered more antibody than directly resuspending the pellets in water (77% versus 61%). Equivalent volumes of the IgY preparation in PBS or water were compared by PAGE, and these results were in accordance with the absorbance values (data not shown).

c) Activity Of IgY Prepared With Different Solvents

An ELISA was performed to compare the binding activity of the IgY extracted by each procedure described above. C. durissus terrificus (C.d.t.) venom at 2.5 μg/ml in PBS was used to coat each well of a 96-well microtiter plate. The remaining protein binding sites were blocked with PBS containing 5 mg/ml BSA. Primary antibody dilutions (in PBS containing 1 mg/ml BSA) were added in duplicate. After 2 hours of incubation at room temperature, the unbound primary antibodies were removed by washing the wells with PBS, BBS-Tween, and PBS. The species specific secondary antibody (goat anti-chicken immunoglobulin alkaline-phosphatase conjugate (Sigma) was diluted 1:750 in PBS containing 1 mg/ml BSA and added to each well of the microtiter plate. After 2 hours of incubation at room temperature, the unbound secondary antibody was removed by washing the plate as before, and freshly prepared alkaline phosphatase substrate (Sigma) at 1 mg/ml in 50 mM Na₂ CO₃, 10 mM MgCl₂, pH 9.5 was added to each well. The color development was measured on a Dynatech MR 700 microplate reader using a 412 nm filter. The results are shown in Table 8.

The binding assay results parallel the recovery values in Table 7, with PBS-dissolved IgY showing slightly more activity than the PBS-dissolved/H₂ O dialyzed antibody. The water-dissolved antibody had considerably less binding activity than the other preparations.

EXAMPLE 5 Survival Of Antibody Activity After Passage Through The Gastrointestinal Tract

In order to determine the feasibility of oral administration of antibody, it was of interest to determine whether orally administered IgY survived passage through the gastrointestinal tract. The example involved: (a) oral administration of specific immune antibody mixed with a nutritional formula; and (b) assay of antibody activity extracted from feces.

                  TABLE 8                                                          ______________________________________                                         Antigen-Binding Activity of IgY Prepared with Different Solvents                                  PBS        H.sub.2 O                                                                               PBS/                                    DILUTION                                                                               PREIMMUNE  DISSOLVED  DISSOLVED                                                                               H.sub.2 O                               ______________________________________                                         1:500   0.005      1.748      1.577    1.742                                   1:2,500 0.004      0.644      0.349    0.606                                   1:12,500                                                                               0.001      0.144      0.054    0.090                                   1:62,500                                                                               0.001      0.025      0.007    0.016                                   1:312,500                                                                              0.010      0.000      0.000    0.002                                   ______________________________________                                    

a) Oral Administration Of Antibody

The IgY preparations used in this example are the same PBS-dissolved/H₂ O dialyzed antivenom materials obtained in Example 4 above, mixed with an equal volume of Enfamil®. Two mice were used in this experiment, each receiving a different diet as follows:

1) water and food as usual;

2) immune IgY preparation dialyzed against water and mixed 1:1 with Enfamil®. (The mice were given the corresponding mixture as their only source of food and water).

b) Antibody Activity After Ingestion

After both mice had ingested their respective fluids, each tube was refilled with approximately 10 ml of the appropriate fluid first thing in the morning. By mid-morning there was about 4 to 5 ml of liquid left in each tube. At this point stool samples were collected from each mouse, weighed, and dissolved in approximately 500 μl PBS per 100 mg stool sample. One hundred and sixty mg of control stools (no antibody) and 99 mg of experimental stools (specific antibody) in 1.5 ml microfuge tubes were dissolved in 800 and 500 gl PBS, respectively. The samples were heated at 37° C. for 10 minutes and vortexed vigorously. The experimental stools were also broken up with a narrow spatula. Each sample was centrifuged for 5 minutes in a microfuge and the supernatants, presumably containing the antibody extracts, were collected. The pellets were saved at 2°-8° C. in case future extracts were needed. Because the supernatants were tinted, they were diluted five-fold in PBS containing 1 mg/ml BSA for the initial dilution in the enzyme immunoassay (ELISA). The primary extracts were then diluted five-fold serially from this initial dilution. The volume of primary extract added to each well was 190 μl. The ELISA was performed exactly as described in Example 4.

                  TABLE 9                                                          ______________________________________                                         Specific Antibody Activity After Passage Through                               the Gastrointestinal Tract                                                                            CONTROL                                                           PREIMMUNE    FECAL     EXP. FECAL                                    DILUTION  IgY          EXTRACT   EXTRACT                                       ______________________________________                                         1:5       <0           0.000     0.032                                         1:25      0.016        <0        0.016                                         1:125     <0           <0        0.009                                         1:625     <0           0.003     0.001                                         1:3125    <0           <0        0.000                                         ______________________________________                                    

There was some active antibody in the fecal extract from the mouse given the specific antibody in Enfamil® formula, but it was present at a very low level. Since the samples were assayed at an initial 1:5 dilution, the binding observed could have been higher with less dilute samples. Consequently, the mice were allowed to continue ingesting either regular food and water or the specific IgY in Enfamil® formula, as appropriate, so the assay could be repeated. Another ELISA plate was coated overnight with 5 μg/ml of C.d.t. venom in PBS.

The following morning the ELISA plate was blocked with 5 mg/ml BSA, and the fecal samples were extracted as before, except that instead of heating the extracts at 37° C., the samples were kept on ice to limit proteolysis. The samples were assayed undiluted initially, and in 5X serial dilutions thereafter. Otherwise the assay was carried out as before.

                  TABLE 10                                                         ______________________________________                                         Specific Antibody Survives Passage Through The Gastrointestinal Tract                   PREIMMUNE   CONTROL                                                   DILUTION IgY         EXTRACT    EXP. EXTRACT                                   ______________________________________                                         undiluted                                                                               0.003       <0         0.379                                          1:5      <0          <0         0.071                                          1:25     0.000       <0         0.027                                          1:125    0.003       <0         0.017                                          1:625    0.000       <0         0.008                                          1:3125   0.002       <0         0.002                                          ______________________________________                                    

The experiment confirmed the previous results, with the antibody activity markedly higher. The control fecal extract showed no anti-C.d.t. activity, even undiluted, while the fecal extract from the anti-C.d.t. IgY/Enfamil®-fed mouse showed considerable anti-C.d.t. activity. This experiment (and the previous experiment) clearly demonstrate that active IgY antibody survives passage through the mouse digestive tract, a finding with favorable implications for the success of IgY antibodies administered orally as a therapeutic or prophylactic.

EXAMPLE 6 In Vivo Neutralization Of Type C. botulinum Type A Neurotoxin By Avian Antitoxin Antibody

This example demonstrated the ability of PEG-purified antitoxin, collected as described in Example 3, to neutralize the lethal effect of C. botulinum neurotoxin type A in mice. To determine the oral lethal dose (LD₁₀₀) of toxin A, groups of BALB/c mice were given different doses of toxin per unit body weight (average body weight of 24 grams). For oral administration, toxin A complex, which contains the neurotoxin associated with other non-toxin proteins was used. This complex is markedly more toxic than purified neurotoxin when given by the oral route. I. Ohishi et al., Infect. Immun., 16:106 (1977).! C. botulinum toxin type A complex, obtained from Eric Johnson (University Of Wisconsin, Madison) was 250 μg/ml in 50 mM sodium citrate, pH 5.5, specific toxicity 3×10⁷ mouse LD₅₀ /mg with parenteral administration. Approximately 40-50 ng/gm body weight was usually fatal within 48 hours in mice maintained on conventional food and water. When mice were given a diet ad libitum of only Enfamil® the concentration needed to produce lethality was approximately 2.5 times higher (125 ng/gm body weight). Botulinal toxin concentrations of approximately 200 ng/gm body weight were fatal in mice fed Enfamil® containing preimmune IgY (resuspended in Enfamil® at the original yolk volume).

The oral LD₁₀₀ of C. botulinum toxin was also determined in mice that received known amounts of a mixture of preimmune IgY-Ensure® delivered orally through feeding needles. Using a 22 gauge feeding needle, mice were given 250 μl each of a preimmune IgY-Ensure® mixture (preimmune IgY dissolved in 1/4 original yolk volume) 1 hour before and 1/2 hour and 5 hours after administering botulinal toxin. Toxin concentrations given orally ranged from approximately 12 to 312 ng/gm body weight (0.3 to 7.5 μg per mouse). Botulinal toxin complex concentration of approximately 40 ng/gm body weight (1 μg per mouse) was lethal in all mice in less than 36 hours.

Two groups of BALB/c mice, 10 per group, were each given orally a single dose of 1 μg each of botulinal toxin complex in 100 μl of 50 mM sodium citrate pH 5.5. The mice received 250 μl treatments of a mixture of either preimmune or immune IgY in Ensure® (1/4 original yolk volume) 1 hour before and 1/2 hour, 4 hours, and 8 hours after botulinal toxin administration. The mice received three treatments per day for two more days. The mice were observed for 96 hours. The survival and mortality are shown in Table 11.

                  TABLE 11                                                         ______________________________________                                         Neutralization Of Botulinal Toxin A In Vivo                                    TOXIN DOSE                                                                              ANTIBODY      NUMBER OF  NUMBER OF                                    ng/gm    TYPE          MICE ALIVE MICE DEAD                                    ______________________________________                                         41.6     non-immune    0          10                                           41.6     anti-botulinal toxin                                                                         10         0                                            ______________________________________                                    

All mice treated with the preimmune IgY-Ensure® mixture died within 46 hours post-toxin administration. The average time of death in the mice was 32 hours post toxin administration. Treatments of preimmune IgY-Ensure® mixture did not continue beyond 24 hours due to extensive paralysis of the mouth in mice of this group. In contrast, all ten mice treated with the immune anti-botulinal toxin IgY-Ensure® mixture survived past 96 hours. Only 4 mice in this group exhibited symptoms of botulism toxicity (two mice about 2 days after and two mice 4 days after toxin administration). These mice eventually died 5 and 6 days later. Six of the mice in this immune group displayed no adverse effects to the toxin and remained alive and healthy long term. Thus, the avian anti-botulinal toxin antibody demonstrated very good protection from the lethal effects of the toxin in the experimental mice.

EXAMPLE 7 Production Of An Avian Antitoxin Against Clostridium difficile Toxin A

Toxin A is a potent cytotoxin secreted by pathogenic strains of C. difficile, that plays a direct role in damaging gastrointestinal tissues. In more severe cases of C. difficile intoxication, pseudomembranous colitis can develop which may be fatal. This would be prevented by neutralizing the effects of this toxin in the gastrointestinal tract. As a first step, antibodies were produced against a portion of the toxin. The example involved: (a) conjugation of a synthetic peptide of toxin A to bovine serum albumin; (b) immunization of hens with the peptide-BSA conjugate; and (c) detection of antitoxin peptide antibodies by ELISA.

a) Conjugation Of A Synthetic Peptide Of Toxin A To Bovine Serum Albumin

The synthetic peptide CQTIDGKKYYFN-NH₂ (SEQ ID NO:31) was prepared commercially (Multiple Peptide Systems, San Diego, Calif.) and validated to be >80% pure by high-pressure liquid chromatography. The eleven amino acids following the cysteine residue represent a consensus sequence of a repeated amino acid sequence found in Toxin A. Wren et al., Infect. Immun., 59:3151-3155 (1991).! The cysteine was added to facilitate conjugation to carrier protein.

In order to prepare the carrier for conjugation, BSA (Sigma) was dissolved in 0.01M NaPO₄, pH 7.0 to a final concentration of 20 mg/ml and n-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Pierce) was dissolved in N,N-dimethyl formamide to a concentration of 5 mg/ml. MBS solution, 0.51 ml, was added to 3.25 ml of the BSA solution and incubated for 30 minutes at room temperature with stirring every 5 minutes. The MBS-activated BSA was then purified by chromatography on a Bio-Gel P-10 column (Bio-Rad; 40 ml bed volume) equilibrated with 50 mM NaPO₄, pH 7.0 buffer. Peak fractions were pooled (6.0 ml).

Lyophitized toxin A peptide (20 mg) was added to the activated BSA mixture, stirred until the peptide dissolved and incubated 3 hours at room temperature. Within 20 minutes, the reaction mixture became cloudy and precipitates formed. After 3 hours, the reaction mixture was centrifuged at 10,000×g for 10 min and the supernatant analyzed for protein content. No significant protein could be detected at 280 nm. The conjugate precipitate was washed three times with PBS and stored at 4° C. A second conjugation was performed with 15 mg of activated BSA and 5 mg of peptide and the conjugates pooled and suspended at a peptide concentration of 10 mg/ml in 10 mM NaPO₄, pH 7.2.

b) Immunization Of Hens With Peptide Conjugate

Two hens were each initially immunized on day zero by injection into two subcutaneous and two intramuscular sites with 1 mg of peptide conjugate that was emulsified in CFA (GIBCO). The hens were boosted on day 14 and day 21 with 1 mg of peptide conjugate emulsified in IFA (GIBCO).

c) Detection Of Antitoxin Peptide Antibodies By ELISA

IgY was purified from two eggs obtained before immunization (pre-immune) and two eggs obtained 31 and 32 days after the initial immunization using PEG fractionation as described in Example 1.

Wells of a 96-well microtiter plate (Falcon Pro-Bind Assay Plate) were coated overnight at 4° C. with 100 μg/ml solution of the toxin A synthetic peptide in PBS, pH 7.2 prepared by dissolving 1 mg of the peptide in 1.0 ml of H₂ O and dilution of PBS. The pre-immune and immune IgY preparations were diluted in a five-fold series in a buffer containing 1% PEG 8000 and 0.1% Tween-20 (v/v) in PBS, pH 7.2. The wells were blocked for 2 hours at room temperature with 150 μl of a solution containing 5% (v/v) Carnation® nonfat dry milk and 1% PEG 8000 in PBS, pH 7.2. After incubation for 2 hours at room temperature, the wells were washed, secondary rabbit anti-chicken IgG-alkaline phosphatase (1:750) added, the wells washed again and the color development obtained as described in Example 1. The results are shown in Table 12.

                  TABLE 12                                                         ______________________________________                                         Reactivity Of IgY With Toxin Peptide                                                         ABSORBANCE AT 410 nm                                                                        IMMUNE ANTI-                                        DILUTION OF PEG PREP                                                                           PREIMMUNE  PEPTIDE                                             ______________________________________                                         1:100           0.013      0.253                                               1:500           0.004      0.039                                                1:2500         0.004      0.005                                               ______________________________________                                    

Clearly, the immune antibodies contain titers against this repeated epitope of toxin A.

EXAMPLE 8 Production Of Avian Antitoxins Against Clostridium difficile Native Toxins A And B

To determine whether avian antibodies are effective for the neutralization of C. difficile toxins, hens were immunized using native C. difficile toxins A and B. The resulting egg yolk antibodies were then extracted and assessed for their ability to neutralize toxins A and B in vitro. The Example involved (a) preparation of the toxin immunogens, (b) immunization, (c) purification of the antitoxins, and (d) assay of toxin neutralization activity.

a) Preparation Of The Toxin Immunogens

Both C. difficile native toxins A and B, and C. difficile toxoids, prepared by the treatment of the native toxins with formaldehyde, were employed as immunogens. C. difficile toxoids A and B were prepared by a procedure which was modified from published methods (Ehrich et al., Infect. Immun. 28:1041 (1980). Separate solutions (in PBS) of native C. difficile toxin A and toxin B (Tech Lab) were each adjusted to a concentration of 0.20 mg/ml, and formaldehyde was added to a final concentration of 0.4%. The toxin/formaldehyde solutions were then incubated at 37° C. for 40 hrs. Free formaldehyde was then removed from the resulting toxoid solutions by dialysis against PBS at 4° C. In previously published reports, this dialysis step was not performed. Therefore, free formaldehyde must have been present in their toxoid preparations. The toxoid solutions were concentrated, using a Centriprep concentrator unit (Amicon), to a final toxoid concentration of 4.0 mg/ml. The two resulting preparations were designated as toxoid A and toxoid B.

C. difficile native toxins were prepared by concentrating stock solutions of toxin A and toxin B (Tech Lab, Inc), using Centriprep concentrator units (Amicon), to a final concentration of 4.0 mg/ml.

b) Immunization

The first two immunizations were performed using the toxoid A and toxoid B immunogens described above. A total of 3 different immunization combinations were employed. For the first immunization group, 0.2 ml of toxoid A was emulsified in an equal volume of Titer Max adjuvant (CytRx). Titer Max was used in order to conserve the amount of immunogen used, and to simplify the immunization procedure. This immunization group was designated "CTA." For the second immunization group, 0.1 ml of toxoid B was emulsified in an equal volume of Titer Max adjuvant. This group was designated "CTB." For the third immunization group, 0.2 ml of toxoid A was first mixed with 0.2 ml of toxoid B, and the resulting mixture was emulsified in 0.4 ml of Titer Max adjuvant. This group was designated "CTAB." In this way, three separate immunogen emulsions were prepared, with each emulsion containing a final concentration of 2.0 mg/ml of toxoid A (CTA) or toxoid B (CTB) or a mixture of 2.0 mg/ml toxoid A and 2.0 mg/ml toxoid B (CTAB).

On day 0, White Leghorn hens, obtained from a local breeder, were immunized as follows: Group CTA. Four hens were immunized, with each hen receiving 200 μg of toxoid A, via two intramuscular (I.M.) injections of 50 μl of CTA emulsion in the breast area. Group CTB. One hen was immunized with 200 μg of toxoid B, via two I.M. injections of 50 μl of CTB emulsion in the breast area. Group CTAB. Four hens were immunized, with each hen receiving a mixture containing 200 μg of toxoid A and 200 μg of toxoid B, via two I.M. injections of 100 μl of CTAB emulsion in the breast area. The second immunization was performed 5 weeks later, on day 35, exactly as described for the first immunization above.

In order to determine whether hens previously immunized with C. difficile toxoids could tolerate subsequent booster immunizations using native toxins, a single hen from group CTAB was immunized for a third time, this time using a mixture of the native toxin A and native toxin B described in section (a) above (these toxins were not formaldehyde-treated, and were used in their active form). This was done in order to increase the amount (titer) and affinity of specific antitoxin antibody produced by the hen over that achieved by immunizing with toxoids only. On day 62, 0.1 ml of a toxin mixture was prepared which contained 200 μg of native toxin A and 200 μg of native toxin B. This toxin mixture was then emulsified in 0.1 ml of Titer Max adjuvant. A single CTAB hen was then immunized with the resulting immunogen emulsion, via two I.M. injections of 100 μl each, into the breast area. This hen was marked with a wing band, and observed for adverse effects for a period of approximately 1 week, after which time the hen appeared to be in good health.

Because the CTAB hen described above tolerated the booster immunization with native toxins A and B with no adverse effects, it was decided to boost the remaining hens with native toxin as well. On day 70, booster immunizations were performed as follows: Group CTA. A 0.2 ml volume of the 4 mg/ml native toxin A solution was emulsified in an equal volume of Titer Max adjuvant. Each of the 4 hens was then immunized with 200 μg of native toxin A, as described for the toxoid A immunizations above. Group CTB. A 50 μl volume of the 4 mg/ml native toxin B solution was emulsified in an equal volume of Titer Max adjuvant. The hen was then immunized with 200 μg of native toxin B, as described for the toxoid B immunizations above. Group CTAB. A 0.15 ml volume of the 4 mg/ml native toxin A solution was first mixed with a 0.15 ml volume the 4 mg/ml native toxin B solution. The resulting toxin mixture was then emulsified in 0.3 ml of Titer Max adjuvant. The 3 remaining hens (the hen with the wing band was not immunized this time) were then immunized with 200 μg of native toxin A and 200 μg of native toxin B as described for the toxoid A+toxoid B immunizations (CTAB) above. On day 85, all hens received a second booster immunization using native toxins, done exactly as described for the first boost with native toxins above.

All hens tolerated both booster immunizations with native toxins with no adverse effects. As previous literature references describe the use of formaldehyde-treated toxoids, this is apparently the first time that any immunizations have been performed using native C. difficile toxins.

c) Purification Of Antitoxins

Eggs were collected from the hen in group CTB 10-12 days following the second immunization with toxoid (day 35 immunization described in section (b) above), and from the hens in groups CTA and CTAB 20-21 days following the second immunization with toxoid. To be used as a pre-immune (negative) control, eggs were also collected from unimmunized hens from the same flock. Egg yolk immunoglobulin (IgY) was extracted from the 4 groups of eggs as described in Example 1 (c), and the final IgY pellets were solubilized in the original yolk volume of PBS without thimerosal. Importantly, thimerosal was excluded because it would have been toxic to the CHO cells used in the toxin neutralization assays described in section (d) below.

d) Assay Of Toxin Neutralization Activity

The toxin neutralization activity of the IgY solutions prepared in section (c) above was determined using an assay system that was modified from published methods. Ehrich et al., Infect. Immun. 28:1041-1043 (1992); and McGee et al. Microb. Path. 12:333-341 (1992).! As additional controls, affinity-purified goat anti-C. difficile toxin A (Tech Lab) and affinity-purified goat anti-C. difficile toxin B (Tech Lab) were also assayed for toxin neutralization activity.

The IgY solutions and goat antibodies were serially diluted using F 12 medium (GIBCO) which was supplemented with 2% FCS (GIBCO)(this solution will be referred to as "medium" for the remainder of this Example). The resulting antibody solutions were then mixed with a standardized concentration of either native C. difficile toxin A (Tech Lab), or native C. difficile toxin B (Tech Lab), at the concentrations indicated below. Following incubation at 37° C. for 60 min., 100 μl volumes of the toxin+antibody mixtures were added to the wells of 96-well microtiter plates (Falcon Microtest III) which contained 2.5×10⁴ Chinese Hamster Ovary (CHO) cells per well (the CHO cells were plated on the previous day to allow them to adhere to the plate wells). The final concentration of toxin, or dilution of antibody indicated below refers to the final test concentration of each reagent present in the respective microtiter plate wells. Toxin reference wells were prepared which contained CHO cells and toxin A or toxin B at the same concentration used for the toxin plus antibody mixtures (these wells contained no antibody). Separate control wells were also prepared which contained CHO cells and medium only. The assay plates were then incubated for 18-24 hrs. in a 37° C., humidified, 5% CO₂ incubator. On the following day, the remaining adherent (viable) cells in the plate wells were stained using 0.2% crystal violet (Mallinckrodt) dissolved in 2% ethanol, for 10 min. Excess stain was then removed by rinsing with water, and the stained cells were solubilized by adding 100 μl of 1% SDS (dissolved in water) to each well. The absorbance of each well was then measured at 570 nm, and the percent cytotoxicity of each test sample or mixture was calculated using the following formula: ##EQU1##

Unlike previous reports which quantitate results visually by counting cell rounding by microscopy, this Example utilized spectrophotometric methods to quantitate the C. difficile toxin bioassay. In order to determine the toxin A neutralizing activity of the CTA, CTAB, and pre-immune IgY preparations, as well as the affinity-purified goat antitoxin A control, dilutions of these antibodies were reacted against a 0.1 μg/ml concentration of native toxin A (this is the approx. 50% cytotoxic dose of toxin A in this assay system). The results are shown in FIG. 3.

Complete neutralization of toxin A occurred with the CTA IgY (antitoxin A, above) at dilutions of 1:80 and lower, while significant neutralization occurred out to the 1:320 dilution. The CTAB IgY (antitoxin A+toxin B, above) demonstrated complete neutralization at the 1:320-1:160 and lower dilutions, and significant neutralization occurred out to the 1:1280 dilution. The commercially available affinity-purified goat antitoxin A did not completely neutralize toxin A at any of the dilutions tested, but demonstrated significant neutralization out to a dilution of 1:1,280. The preimmune IgY did not show any toxin A neutralizing activity at any of the concentrations tested. These results demonstrate that IgY purified from eggs laid by hens immunized with toxin A alone, or simultaneously with toxin A and toxin B, is an effective toxin A antitoxin.

The toxin B neutralizing activity of the CTAB and pre-immune IgY preparations, and also the affinity-purified goat antitoxin B control was determined by reacting dilutions of these antibodies against a concentration of native toxin B of 0.1 ng/ml (approximately the 50% cytotoxic dose of toxin B in the assay system). The results are shown in FIG. 4.

Complete neutralization of toxin B occurred with the CTAB IgY (antitoxin A+toxin B, above) at the 1:40 and lower dilutions, and significant neutralization occurred out to the 1:320 dilution. The affinity-purified goat antitoxin B demonstrated complete neutralization at dilutions of 1:640 and lower, and significant neutralization occurred out to a dilution of 1:2,560. The preimmune IgY did not show any toxin B neutralizing activity at any of the concentrations tested. These results demonstrate that IgY purified from eggs laid by hens immunized simultaneously with toxin A and toxin B is an effective toxin B antitoxin.

In a separate study, the toxin B neutralizing activity of CTB, CTAB, and preimmune IgY preparations was determined by reacting dilutions of these antibodies against a native toxin B concentration of 0.1 μg/ml (approximately 100% cytotoxic dose of toxin B in this assay system). The results are shown in FIG. 5.

Significant neutralization of toxin B occurred with the CTB IgY (antitoxin B, above) at dilutions of 1:80 and lower, while the CTAB IgY (antitoxin A+toxin B, above) was found to have significant neutralizing activity at dilutions of 1:40 and lower. The preimmune IgY did not show any toxin B neutralizing activity at any of the concentrations tested. These results demonstrate that IgY purified from eggs laid by hens immunized with toxin B alone, or simultaneously with toxin A and toxin B, is an effective toxin B antitoxin.

EXAMPLE 9 In vivo Protection Of Golden Syrian Hamsters From C. difficile Disease By Avian Antitoxins Against C. difficile Toxins A And B

The most extensively used animal model to study C. difficile disease is the hamster. Lyerly et al., Infect. Immun. 47:349-352 (1992).! Several other animal models for antibiotic-induced diarrhea exist, but none mimic the human form of the disease as closely as the hamster model. R. Fekety, "Animal Models of Antibiotic-Induced Colitis," in O. Zak and M. Sande (eds.), Experimental Models in Antimicrobial Chemotherapy, Vol. 2, pp. 61-72, (1986).! In this model, the animals are first predisposed to the disease by the oral administration of an antibiotic, such as clindamycin, which alters the population of normally-occurring gastrointestinal flora (Fekety, at 61-72). Following the oral challenge of these animals with viable C. difficile organisms, the hamsters develop cecitis, and hemorrhage, ulceration, and inflammation are evident in the intestinal mucosa. Lyerly et al., Infect. Immun. 47:349-352 (1985).! The animals become lethargic, develop severe diarrhea, and a high percentage of them die from the disease. Lyerly et al., Infect. Immun. 47:349-352 (1985).! This model is therefore ideally suited for the evaluation of therapeutic agents designed for the treatment or prophylaxis of C. difficile disease.

The ability of the avian C. difficile antitoxins, described in Example 1 above, to protect hamsters from C. difficile disease was evaluated using the Golden Syrian hamster model of C. difficile infection. The Example involved (a) preparation of the avian C. difficile antitoxins, (b) in vivo protection of hamsters from C. difficile disease by treatment with avian antitoxins, and (c) long-term survival of treated hamsters.

a) Preparation Of The Avian C. difficile Antitoxins

Eggs were collected from hens in groups CTA and CTAB described in Example 1 (b) above. To be used as a pre-immune (negative) control, eggs were also purchased from a local supermarket. Egg yolk immunoglobulin (IgY) was extracted from the 3 groups of eggs as described in Example 1 (c), and the final IgY pellets were solubilized in one fourth the original yolk volume of Ensure® nutritional formula.

b) In vivo Protection Of Hamsters Against C. difficile Disease By Treatment With Avian Antitoxins

The avian C. difficile antitoxins prepared in section (a) above were evaluated for their ability to protect hamsters from C. difficile disease using an animal model system which was modified from published procedures. Fekety, at 61-72; Borriello et al., J. Med. Microbiol., 24:53-64 (1987); Kim et al., Infect. Immun., 55:2984-2992 (1987); Borriello et al., J. Med. Microbiol., 25:191-196 (1988); Delmee and Avesani, J. Med. Microbiol., 33:85-90 (1990); and Lyerly et al., Infect. Immun., 59:2215-2218 (1991).! For the study, three separate experimental groups were used, with each group consisting of 7 female Golden Syrian hamsters (Charles River), approximately 10 weeks old and weighing approximately 100 gms. each. The three groups were designated "CTA," "CTAB" and "Pre-immune." These designations corresponded to the antitoxin preparations with which the animals in each group were treated. Each animal was housed in an individual cage, and was offered food and water ad libitum through the entire length of the study. On day 1, each animal was orally administered 1.0 ml of one of the three antitoxin preparations (prepared in section (a) above) at the following timepoints: 0 hrs., 4 hrs., and 8 hrs. On day 2, the day 1 treatment was repeated. On day 3, at the 0 hr. timepoint, each animal was again administered antitoxin, as described above. At 1 hr., each animal was orally administered 3.0 mg of clindamycin-HCl (Sigma) in 1 ml of water. This treatment predisposed the animals to infection with C. difficile. As a control for possible endogenous C. difficile colonization, an additional animal from the same shipment (untreated) was also administered 3.0 mg of clindamycin-HCl in the same manner. This clindamycin control animal was left untreated (and uninfected) for the remainder of the study. At the 4 hr. and 8 hr. timepoints, the animals were administered antitoxin as described above. On day 4, at the 0 hr. timepoint, each animal was again administered antitoxin as described above. At 1 hr., each animal was orally challenged with 1 ml of C. difficile inoculum, which contained approx. 100 C. difficile strain 43596 organisms in sterile saline. C. difficile strain 43596, which is a serogroup C strain, was chosen because it is representative of one of the most frequently-occurring serogroups isolated from patients with antibiotic-associated pseudomembranous colitis. Delmee et al., J. Clin. Microbiol., 28:2210-2214 (1985).! In addition, this strain has been previously demonstrated to be virulent in the hamster model of infection. Delmee and Avesani, J. Med. Microbiol., 33:85-90 (1990).! At the 4 hr. and 8 hr. timepoints, the animals were administered antitoxin as described above. On days 5 through 13, the animals were administered antitoxin 3× per day as described for day 1 above, and observed for the onset of diarrhea and death. On the morning of day 14, the final results of the study were tabulated. These results are shown in Table 13.

Representative animals from those that died in the Pre-Immune and CTA groups were necropsied. Viable C. difficile organisms were cultured from the ceca of these animals, and the gross pathology of the gastrointestinal tracts of these animals was consistent with that expected for C. difficile disease (inflamed, distended, hemorrhagic cecum, filled with watery diarrhea-like material). In addition, the

                  TABLE 13                                                         ______________________________________                                         Treatment Results                                                                                No. Animals                                                  Treatment Group   Surviving No. Animals Dead                                   ______________________________________                                         Pre-Immune        1         6                                                  CTA (Antitoxin A only)                                                                           5         2                                                  CTAB (Antitoxin A + Antitoxin B)                                                                 7         0                                                  ______________________________________                                    

clindamycin control animal remained healthy throughout the entire study period, therefore indicating that the hamsters used in the study had not previously been colonized with endogenous C. difficile organisms prior to the start of the study. Following the final antitoxin treatment on day 13, a single surviving animal from the CTA group, and also from the CTAB group, was sacrificed and necropsied. No pathology was noted in either animal.

Treatment of hamsters with orally-administered toxin A and toxin B antitoxin (group CTAB) successfully protected 7 out of 7 (100%) of the animals from C. difficile disease. Treatment of hamsters with orally-administered toxin A antitoxin (group CTA) protected 5 out of 7 (71%) of these animals from C. difficile disease. Treatment using pre-immune IgY was not protective against C. difficile disease, as only 1 out of 7 (14%) of these animals survived. These results demonstrate that the avian toxin A antitoxin and the avian toxin A+toxin B antitoxin effectively protected the hamsters from C. difficile disease. These results also suggest that although the neutralization of toxin A alone confers some degree of protection against C. difficile disease, in order to achieve maximal protection, simultaneous antitoxin A and antitoxin B activity is necessary.

c) Long-Term Survival Of Treated Hamsters

It has been previously reported in the literature that hamsters treated with orally-administered bovine antitoxin IgG concentrate are protected from C. difficile disease as long as the treatment is continued, but when the treatment is stopped, the animals develop diarrhea and subsequently die within 72 hrs. Lyerly et al., Infect. Immun., 59(6):2215-2218 (1991).!

In order to determine whether treatment of C. difficile disease using avian antitoxins promotes long-term survival following the discontinuation of treatment, the 4 surviving animals in group CTA, and the 6 surviving animals in group CTAB were observed for a period of 11 days (264 hrs.) following the discontinuation of antitoxin treatment described in section (b) above. All hamsters remained healthy through the entire post-treatment period. This result demonstrates that not only does treatment with avian antitoxin protect against the onset of C. difficile disease (i.e., it is effective as a prophylactic), it also promotes long-term survival beyond the treatment period, and thus provides a lasting cure.

EXAMPLE 10 In vivo Treatment Of Established C. difficile Infection In Golden Syrian Hamsters With Avian Antitoxins Against C. difficile Toxins A And B

The ability of the avian C. difficile antitoxins, described in Example 8 above, to treat an established C. difficile infection was evaluated using the Golden Syrian hamster model. The Example involved (a) preparation of the avian C. difficile antitoxins, (b) in vivo treatment of hamsters with established C. difficile infection, and (c) histologic evaluation of cecal tissue.

a) Preparation Of The Avian C. difficile Antitoxins

Eggs were collected from hens in group CTAB described in Example 8 (b) above, which were immunized with C. difficile toxoids and native toxins A and B. Eggs purchased from a local supermarket were used as a pre-immune (negative) control. Egg yolk immunoglobulin (IgY) was extracted from the 2 groups of eggs as described in Example 1 (c), and the final IgY pellets were solubilized in one-fourth the original yolk volume of Ensure® nutritional formula.

b) In vivo Treatment Of Hamsters With Established C. difficile Infection

The avian C. difficile antitoxins prepared in section (a) above were evaluated for the ability to treat established C. difficile infection in hamsters using an animal model system which was modified from the procedure which was described for the hamster protection study in Example 8(b) above.

For the study, four separate experimental groups were used, with each group consisting of 7 female Golden Syrian hamsters (Charles River), approx. 10 weeks old, weighing approximately 100 gms. each. Each animal was housed separately, and was offered food and water ad libitum through the entire length of the study.

On day 1 of the study, the animals in all four groups were each predisposed to C. difficile infection by the oral administration of 3.0 mg of clindamycin-HCl (Sigma) in 1 ml of water.

On day 2, each animal in all four groups was orally challenged with 1 ml of C. difficile inoculum, which contained approximately 100 C. difficile strain 43596 organisms in sterile saline. C. difficile strain 43596 was chosen because it is representative of one of the most frequently-occurring serogroups isolated from patients with antibiotic-associated pseudomembranous colitis. Delmee et al., J. Clin. Microbiol., 28:2210-2214 (1990).! In addition, as this was the same C. difficile strain used in all of the previous Examples above, it was again used in order to provide experimental continuity.

On day 3 of the study (24 hrs. post-infection), treatment was started for two of the four groups of animals. Each animal of one group was orally administered 1.0 ml of the CTAB IgY preparation (prepared in section (a) above) at the following timepoints: 0 hrs., 4 hrs., and 8 hrs. The animals in this group were designated "CTAB-24." The animals in the second group were each orally administered 1.0 ml of the pre-immune IgY preparation (also prepared in section (a) above) at the same timepoints as for the CTAB group. These animals were designated "Pre-24." Nothing was done to the remaining two groups of animals on day 3.

On day 4, 48 hrs. post-infection, the treatment described for day 3 above was repeated for the CTAB-24 and Pre-24 groups, and was initiated for the remaining two groups at the same timepoints. The final two groups of animals were designated "CTAB-48" and "Pre-48" respectively.

On days 5 through 9, the animals in all four groups were administered antitoxin or pre-immune IgY, 3× per day, as described for day 4 above. The four experimental groups are summarized in Table 14.

                  TABLE 14                                                         ______________________________________                                         Experimental Treatment Groups                                                  Group Designation                                                                         Experimental Treatment                                              ______________________________________                                         CTAB-24    Infected, treatment w/antitoxin IgY started @ 24                               hrs. post-infection.                                                Pre-24     Infected, treatment w/pre-immune IgY started @ 24                              hrs. post-infection.                                                CTAB-48    Infected, treatment w/antitoxin IgY started @ 48                               hrs. post-infection.                                                Pre-48     Infected, treatment w/pre-immune IgY started @ 48                              hrs. post-infection.                                                ______________________________________                                    

All animals were observed for the onset of diarrhea and death through the conclusion of the study on the morning of day 10. The results of this study are displayed in Table 15.

Eighty-six percent of the animals which began receiving treatment with antitoxin IgY at 24 hrs. post-infection (CTAB-24 above) survived, while 57% of the animals treated with antitoxin IgY starting 48 hrs. post-infection (CTAB-48 above) survived. In contrast, none of the animals receiving pre-immune IgY starting 24 hrs. post-infection (Pre-24 above) survived, and only 29% of the animals which began receiving treatment with pre-immune IgY at 48 hrs. post-infection (Pre-48 above) survived through the conclusion of the study. These results demonstrate that avian antitoxins raised against C. difficile toxins A and B are capable of successfully treating established C. difficile infections in vivo.

                  TABLE 15                                                         ______________________________________                                         Experimental Outcome-Day 10                                                    Treatment Group                                                                             No. Animals Surviving                                                                        No. Animals Dead                                    ______________________________________                                         CTAB-24      6             1                                                   Pre-24       0             7                                                   CTAB-48      4             3                                                   Pre-48       2             5                                                   ______________________________________                                    

c) Histologic Evaluation Of Cecal Tissue

In order to father evaluate the ability of the IgY preparations tested in this study to treat established C. difficile infection, histologic evaluations were performed on cecal tissue specimens obtained from representative animals from the study described in section (b) above.

Immediately following death, cecal tissue specimens were removed from animals which died in the Pre-24 and Pre-48 groups. Following the completion of the study, a representative surviving animal was sacrificed and cecal tissue specimens were removed from the CTAB-24 and CTAB-48 groups. A single untreated animal from the same shipment as those used in the study was also sacrificed and a cecal tissue specimen was removed as a normal control. All tissue specimens were fixed overnight at 4° C. in 10% buffered formalin. The fixed tissues were paraffin-embedded, sectioned, and mounted on glass microscope slides. The tissue sections were then stained using hematoxylin and eosin (H and E stain), and were examined by light microscopy.

Upon examination, the tissues obtained from the CTAB-24 and CTAB-48 animals showed no pathology, and were indistinguishable from the normal control. This observation provides further evidence for the ability of avian antitoxins raised against C. difficile toxins A and B to effectively treat established C. difficile infection, and to prevent the pathologic consequences which normally occur as a result of C. difficile disease.

In contrast, characteristic substantial mucosal damage and destruction was observed in the tissues of the animals from the Pre-24 and Pre-48 groups which died from C. difficile disease. Normal tissue architecture was obliterated in these two preparations, as most of the mucosal layer was observed to have sloughed away, and there were numerous large hemorrhagic areas containing massive numbers of erythrocytes.

EXAMPLE 11 Cloning And Expression Of C. difficile Toxin A Fragments

The toxin A gene has been cloned and sequenced, and shown to encode a protein of predicted MW of 308 kd. Dove et al., Infect. Immun., 58:480-488 (1990).! Given the expense and difficulty of isolating native toxin A protein, it would be advantageous to use simple and inexpensive procaryotic expression systems to produce and purify high levels of recombinant toxin A protein for immunization purposes. Ideally, the isolated recombinant protein would be soluble in order to preserve native antigenicity, since solubilized inclusion body proteins often do not fold into native conformations. To allow ease of purification, the recombinant protein should be expressed to levels greater than 1 mg/liter of E. coli culture.

To determine whether high levels of recombinant toxin A protein can be produced in E. coli, fragments of the toxin A gene were cloned into various prokaryotic expression vectors, and assessed for the ability to express recombinant toxin A protein in E. coli. Three prokaryotic expression systems were utilized. These systems were chosen because they drive expression of either fusion (pMALc and pGEX2T) or native (pET23a-c) protein to high levels in E. coli, and allow affinity purification of the expressed protein on a ligand containing column. Fusion proteins expressed from pGEX vectors bind glutathione agarose beads, and are eluted with reduced glutathione. pMAL fusion proteins bind amylose resin, and are eluted with maltose. A poly-histidine tag is present at either the N-terminal (pET16b) or C-terminal (pET23a-c) end of pET fusion proteins. This sequence specifically binds Ni₂ ⁺ chelate columns, and is eluted with imidazole salts. Extensive descriptions of these vectors are available (Williams et al. (1994) DNA Cloning: Expression Systems, in press), and will not be discussed in detail here. The Example involved (a) cloning of the toxin A gene, (b) expression of large fragments of toxin A in various prokaryotic expression systems, (c) identification of smaller toxin A gene fragments that express efficiently in E. coli, (d) purification of recombinant toxin A protein by affinity chromatography, and (e) demonstration of functional activity of a recombinant fragment of the toxin A gene.

a) Cloning Of The Toxin A Gene

A restriction map of the toxin A gene is shown in FIG. 6. The encoded protein contains a carboxy terminal ligand binding region, containing multiple repeats of a carbohydrate binding domain. von Eichel-Streiber and Sauerborn, Gene 96:107-113 (1990).! The toxin A gene was cloned in three pieces, by using either the polymerase chain reaction (PCR) to amplify specific regions, (regions 1 and 2, FIG. 6) or by screening a constructed genomic library for a specific toxin A gene fragment (region 3, FIG. 6). The sequences of the utilized PCR primers are P1: 5' GGAAATT TAGCTGCAGCATCTGAC 3' (SEQ ID NO:1); P2: 5' TCTAGCAAATTCGCTTGT GTTGAA 3' (SEQ ID NO:2); P3: 5' CTCGCATATAGCATTAGACC 3' (SEQ ID NO:3); and P4: 5' CTATCTAGGCCTAAAGTAT 3' (SEQ ID NO:4). These regions were cloned into prokaryotic expression vectors that express either fusion (pMALc and pGEX2T) or native (pET23a-c) protein to high levels in E. coli, and allow affinity purification of the expressed protein on a ligand containing column.

Clostridium difficile VPI strain 10463 was obtained from the ATCC (ATCC #43255) and grown under anaerobic conditions in brain-heart infusion medium (BBL). High molecular-weight C. difficile DNA was isolated essentially as described by Wren and Tabaqchali (1987) J. Clin. Microbiol., 25:2402, except proteinase K and sodium dodecyl sulfate (SDS) was used to disrupt the bacteria, and cetyltrimethylammonium bromide precipitation as described in Ausubel et al., Current Protocols in Molecular Biology (1989)! was used to remove carbohydrates from the cleared lysate. The integrity and yield of genomic DNA was assessed by comparison with a serial dilution of uncut lambda DNA after electrophoresis on an agarose gel.

Fragments 1 and 2 were cloned by PCR, utilizing a proofreading thermostable DNA polymerase (native pfu polymerase; Stratagene). The high fidelity of this polymerase reduces the mutation problems associated with amplification by error prone polymerases (e.g., Taq polymerase). PCR amplification was performed using the indicated PCR primers (FIG. 6) in 50 μl reactions containing 10 mM Tris-HCl(8.3), 50 mM KCl, 1.5 mM MgCl₂, 200 μM each dNTP, 0.2 μM each primer, and 50 ng C. difficile genomic DNA. Reactions were overlaid with 100 μl mineral oil, heated to 94° C. for 4 min, 0.5 μl native pfu polymerase (Stratagene) added, and the reaction cycled 30× at 94° C. for 1 min, 50° C. for 1 min, 72° C. for 4 min, followed by 10 min at 72° C. Duplicate reactions were pooled, chloroform extracted, and ethanol precipitated. After washing in 70% ethanol, the pellets were resuspended in 50 μl TE buffer 10 mM Tris-HCL, 1 mM EDTA pH 8.0!. Aliquots of 10 μl each were restriction digested with either EcoRI/HincII (fragment 1) or EcoRI/PstI (fragment 2), and the appropriate restriction fragments were gel purified using the Prep-A-Gene kit (BioRad), and ligated to either EcoRI/SmaI-restricted pGEX2T (Pharmacia) vector (fragment 1), or the EcoRI/PstI pMAlc (New England Biolabs) vector (fragment 2). Both clones are predicted to produce in-frame fusions with either the glutathione-S-transferase protein (pGEX vector) or the maltose binding protein (pMAL vector). Recombinant clones were isolated, and confirmed by restriction digestion, using standard recombinant molecular biology techniques. Sambrook et al., Molecular Cloning, A Laboratory Manual (1989), and designated pGA30-660 and pMA660-1100, respectively (see FIG. 6 for description of the clone designations).!

Fragment 3 was cloned from a genomic library of size selected PstI digested C. difficile genomic DNA, using standard molecular biology techniques (Sambrook et al.). Given that the fragment 3 internal PstI site is protected from cleavage in C. difficile genomic DNA Price et al., Curr. Microbiol., 16:55-60 (1987)!, a 4.7 kb fragment from PstI restricted C. difficile genomic DNA was gel purified, and ligated to PstI restricted, phosphatase treated pUC9 DNA. The resulting genomic library was screened with a oligonucleotide primer specific to fragment 3, and multiple independent clones were isolated. The presence of fragment 3 in several of these clones was confirmed by restriction digestion, and a clone of the indicated orientation (FIG. 6) was restricted with BamHI/HindIII, the released fragment purified by gel electrophoresis, and ligated into similarly restricted pET23c expression vector DNA (Novagert). Recombinant clones were isolated, and confirmed by restriction digestion. This construct is predicted to create both a predicted in frame fusion with the pET protein leader sequence, as well as a predicted C-terminal poly-histidine affinity tag, and is designated pPA1100-2680 (see FIG. 6 for the clone designation).

b) Expression Of Large Fragments Of Toxin A In E. coli

Protein expression from the three expression constructs made in (a) was induced, and analyzed by Western blot analysis with an affinity purified, goat polyclonal antiserum directed against the toxin A toxoid (Tech Lab). The procedures utilized for protein induction, SDS-PAGE, and Western blot analysis are described in detail in Williams et al (1994), supra. In brief, 5 ml 2X YT (16 g tryptone, 10 g yeast extract, 5 g NaCl per liter, pH 7.5+100 μg/ml ampicillin were added to cultures of bacteria (BL21 for pMAl and pGEX plasmids, and BL21 (DE3)LysS for pET plasmids) containing the appropriate recombinant clone which were induced to express recombinant protein by addition of IPTG to 1 mM. Cultures were grown at 37° C., and induced when the cell density reached 0.5 OD₆₀₀. Induced protein was allowed to accumulate for two hrs after induction. Protein samples were prepared by pelleting 1 ml aliquots of bacteria by centrifugation (1 min in a microfuge), and resuspension of the pelleted bacteria in 150 μl of 2× SDS-PAGE sample buffer Williams et al. (1994), supra!. The samples were heated to 95° C. for 5 min, the cooled and 5 or 10 μl aliquots loaded on 7.5% SDS-PAGE gels. BioRad high molecular weight protein markers were also loaded, to allow estimation of the MW of identified fusion proteins. After electrophoresis, protein was detected either generally by staining gels with Coomassie blue, or specifically, by blotting to nitrocellulose for Western blot detection of specific immunoreactive protein. Western blots, (performed as described in Example 3) which detect toxin A reactive protein in cell lysates of induced protein from the three expression constructs are shown in FIG. 7. In this figure, lanes 1-3 contain cell lysates prepared from E. coli strains containing pPA1100-2860 in B121(DE3)lysE cells; lanes 4-6 contain cell lysates prepared from E. coli strains containing pPA1100-2860 in B121(DE3)lysS cells; lanes 7-9 contain cell lysates prepared from E. coli strains containing pMA30-660; lanes 10-12 contain cell lysates prepared from E. coli strains containing pMA660-1100. The lanes were probed with an affinity purified goat antitoxin A polyclonal antibody (Tech Lab). Control lysates from uninduced cells (lanes 1, 7, and 10) contain very little immunoreactive material compared to the induced samples in the remaining lanes. The highest molecular weight band observed for each clone is consistent with the predicted size of the full length fusion protein.

Each construct directs expression of high molecular weight (HMW) protein that is reactive with the toxin A antibody. The size of the largest immunoreactive bands from each sample is consistent with predictions of the estimated MW of the intact fusion proteins. This demonstrates that the three fusions are in-frame, and that none of the clones contain cloning artifacts that disrupt the integrity of the encoded fusion protein. However, the Western blot demonstrates that fusion protein from the two larger constructs (pGA30-660 and pPA1100-2680) are highly degraded. Also, expression levels of toxin A proteins from these two constructs are low, since induced protein bands are not visible by Coomassie staining (not shown). Several other expression constructs that fuse large sub-regions of the toxin A gene to either pMALc or pET23a-c expression vectors, were constructed and tested for protein induction. These constructs were made by mixing gel purified restriction fragments, derived from the expression constructs shown in FIG. 6, with appropriately cleaved expression vectors, ligating, and selecting recombinant clones in which the toxin A restriction fragments had ligated together and into the expression vector as predicted for in-frame fusions. The expressed toxin A interval within these constructs are shown in FIG. 8, as well as the internal restriction sites utilized to make these constructs.

As used herein, the term "interval" refers to any portion (i.e., any segment of the toxin which is less than the whole toxin molecule) of a clostridial toxin. In a preferred embodiment, "interval" refers to portions of C. difficile toxins such as toxin A or toxin B. It is also contemplated that these intervals will correspond to epitopes of immunologic importance, such as antigens or immunogens against which a neutralizing antibody response is effected. It is not intended that the present invention be limited to the particular intervals or sequences described in these Examples. It is also contemplated that sub-portions of intervals (e.g., an epitope contained within one interval or which bridges multiple intervals) be used as compositions and in the methods of the present invention.

In all cases, Western blot analysis of each of these constructs with goat antitoxin A antibody (Tech Lab) detected HMW fusion protein of the predicted size (not shown). This confirms that the reading frame of each of these clones is not prematurely terminated, and is fused in the correct frame with the fusion partner. However, the Western blot analysis revealed that in all cases, the induced protein is highly degraded, and, as assessed by the absence of identifiable induced protein bands by Coomassie Blue staining, are expressed only at low levels. These results suggest that expression of high levels of intact toxin A recombinant protein is not possible when large regions of the toxin A gene are expressed in E. coli using these expression vectors.

c) High Level Expression Of Small Toxin A Protein Fusions In E. coli

Experience indicates that expression difficulties are often encountered when large (greater than 100 kd) fragments are expressed in E. coli. A number of expression constructs containing smaller fragments of the toxin A gene were constructed, to determine if small regions of the gene can be expressed to high levels without extensive protein degradation. A summary of these expression constructs are shown in FIG. 9. All were constructed by in-frame fusions of convenient toxin A restriction fragments to either the pMALc or pET23a-c vectors. Protein preparations from induced cultures of each of these constructs were analyzed by both Coomassie Blue staining and Western analysis as in (b) above. In all cases, higher levels of intact, full length fusion proteins were observed than with the larger recombinants from section (b).

d) Purification Of Recombinant Toxin A Protein

Large scale (500 ml) cultures of each recombinant from (c) were grown, induced, and soluble and insoluble protein fractions were isolated. The soluble protein extracts were affinity chromatographed to isolate recombinant fusion protein, as described Williams et al. (1994), supra!. In brief, extracts containing tagged pET fusions were chromatographed on a nickel chelate column, and eluted using imidazole salts as described by the distributor (Novagen). Extracts containing soluble pMAL fusion protein were prepared and chromatographed in column buffer (10 mM NaPO4, 0.5M NaCl, 10 mM β-mercaptoethanol, pH 7.2) over an amylose resin column (New England Biolabs), and eluted with column buffer containing 10 mM maltose as described Williams et al. (1994), supra!. When the expressed protein was found to be predominantly insoluble, insoluble protein extracts were prepared by the method described in Example 17, infra. The results are summarized in Table 16. FIG. 10 shows the sample purifications of recombinant toxin A protein. In this figure, lanes 1 and 2 contain MBP fusion protein purified by affinity purification of soluble protein.

                                      TABLE 16                                     __________________________________________________________________________     Purification Of Recombinant Toxin A Protein                                                            % Intact                                                              Yield Affinity                                                                          Soluble                                                                               Yield Intact                                            Protein                                                                               Purified Soluble                                                                        Fusion Insoluble                                       Clone.sup.(a)                                                                          Solubuility                                                                           Protein.sup.(b)                                                                         Protein.sup.(c)                                                                       Fusion Protein                                  __________________________________________________________________________     pMA30-270                                                                              Soluble                                                                               4 mg/500 mls                                                                            10%    NA                                              PMA30-300                                                                              Soluble                                                                               4 mg/500 mls                                                                            5-10%  NA                                              pMA300-660                                                                             Insoluble                                                                             --       NA     10 mg/500 ml                                    pMA660- Soluble                                                                               4.5 mg/500 mls                                                                          50%    NA                                              1100                                                                           pMA1100-                                                                               Soluble                                                                               18 mg/500 mls                                                                           10%    NA                                              1610                                                                           pMA1610-                                                                               Both   22 mg/500 mls                                                                           90%    20 mg/500 ml                                    1870                                                                           pMA1450-                                                                               Insoluble                                                                             --       NA     0.2 mg/500 ml                                   1870                                                                           pPA1100-                                                                               Soluble                                                                               0.1 mg/500 mls                                                                          90%    NA                                              1450                                                                           pPA1100-                                                                               Soluble                                                                               0.02 mg/500                                                                             90%    NA                                              1870           mls                                                             pMA1870-                                                                               Both   12 mg/500 mls                                                                           80%    NA                                              2680                                                                           pPA1870-                                                                               Insoluble                                                                             --       NA     10 mg/500 ml                                    2680                                                                           __________________________________________________________________________      .sup.(a) pP = pET23 vector, pM = pMALc vector, A = toxin A.                    .sup.(b) Based on 1.5 OD.sub.280 = 1 mg/ml (extinction coefficient of          MBP).                                                                          .sup.(c) Estimated by Coomassie staining of SDSPAGE gels.                

Lanes 3 and 4 contain MBP fusion protein purified by solubilization of insoluble inclusion bodies. The purified fusion protein samples are pMA1870-2680 (lane 1), pMA660-1100 (lane 2), pMA300-600 (lane 3) and pMA1450-1870 (lane 4).

Poor yields of affinity purified protein were obtained when poly-histidine tagged pET vectors were used to drive expression (pPA1100-1450, pP1100-1870). However, significant protein yields were obtained from pMAL expression constructs spanning the entire toxin A gene, and yields of full-length soluble fusion protein ranged from an estimated 200-400 μg/500 ml culture (pMA30-300) to greater than 20 mg/500 ml culture (pMA1610-1870). Only one interval was expressed to high levels as strictly insoluble protein (pMA300-660). Thus, although high level expression was not observed when using large expression constructs from the toxin A gene, usable levels of recombinant protein spanning the entire toxin A gene were obtainable by isolating induced protein from a series of smaller pMAL expression constructs that span the entire toxin A gene. This is the first demonstration of the feasibility of expressing recombinant toxin A protein to high levels in E. coli.

e) Hemagglutination Assay Using The Toxin A Recombinant Proteins

The carboxy terminal end consisting of the repeating units contains the hemagglutination activity or binding domain of C. difficile toxin A. To determine whether the expressed toxin A recombinants retain functional activity, hemagglutination assays were performed. Two toxin A recombinant proteins, one containing the binding domain as either soluble affinity purified protein (pMA1870-2680) or SDS solubilized inclusion body protein (pPA1870-2680) and soluble protein from one region outside that domain (pMA1100-1610) were tested using a described procedure. H. C. Krivan el. al., Infect. Immun., 53:573 (1986).! Citrated rabbit red blood cells (RRBC)(Cocalico) were washed several times with Tris-buffer (0.1M Tris and 50 mM NaCl) by centrifugation at 450×g for 10 minutes at 4° C. A 1% RRBC suspension was made from the packed cells and resuspended in Tris-buffer. Dilutions of the recombinant proteins and native toxin A (Tech Labs) were made in the Tris-buffer and added in duplicate to a round-bottomed 96-well microtiter plate in a final volume of 100 μl. To each well, 50 μl of the 1% RRBC suspension was added, mixed by gentle tapping, and incubated at 4° C. for 3-4 hours. Significant hemagglutination occurred only in the recombinant proteins containing the binding domain (pMA 1870-2680) and native toxin A. The recombinant protein outside the binding domain (pMA 1100-1610) displayed no hemagglutination activity. Using equivalent protein concentrations, the hemagglutination titer for toxin A was 1:256, while titers for the soluble and insoluble recombinant proteins of the binding domain were 1:256 and about 1:5000. Clearly, the recombinant proteins tested retained functional activity and were able to bind RRBC's.

EXAMPLE 12 Functional Activity Of IgY Reactive Against Toxin A Recombinants

The expression of recombinant toxin A protein as multiple fragments in E. coli has demonstrated the feasibility of generating toxin A antigen through use of recombinant methodologies (Example 11). The isolation of these recombinant proteins allows the immunoreactivity of each individual subregion of the toxin A protein to be determined (i.e., in a antibody pool directed against the native toxin A protein). This identifies the regions (if any) for which little or no antibody response is elicited when the whole protein is used as a immunogen. Antibodies directed against specific fragments of the toxin A protein can be purified by affinity chromatography against recombinant toxin A protein, and tested for neutralization ability. This identifies any toxin A subregions that are essential for producing neutralizing antibodies. Comparison with the levels of immune response directed against these intervals when native toxin is used as an immunogen predicts whether potentially higher titers of neutralizing antibodies can be produced by using recombinant protein directed against a individual region, rather than the entire protein. Finally, since it is unknown whether antibodies reactive to the recombinant toxin A proteins produced in Example 11 neutralize toxin A as effectively as antibodies raised against native toxin A (Examples 9 and 10), the protective ability of a pool of antibodies affinity purified against recombinant toxin A fragments was assessed for its ability to neutralize toxin A.

This Example involved (a) epitope mapping of the toxin A protein to determine the titre of specific antibodies directed against individual subregions of the toxin A protein when native toxin A protein is used as an immunogen, (b) affinity purification of IgY reactive against recombinant proteins spanning the toxin A gene, (c) toxin A neutralization assays with affinity purified IgY reactive to recombinant toxin A protein to identify subregions of the toxin A protein that induce the production of neutralizing antibodies, and determination of whether complete neutralization of toxin A can be elicited with a mixture of antibodies reactive to recombinant toxin A protein.

a) Epitope Mapping Of The Toxin A Gene

The affinity purification of recombinant toxin A protein specific to defined intervals of the toxin A protein allows epitope mapping of antibody pools directed against native toxin A. This has not previously been possible, since previous expression of toxin A recombinants has been assessed only by Western blot analysis, without knowledge of the expression levels of the protein e.g., von Eichel-Streiber et al, J. Gen. Microbiol., 135:55-64 (1989)!. Thus, high or low reactivity of recombinant toxin A protein on Western blots may reflect protein expression level differences, not immunoreactivity differences. Given that the purified recombinant protein generated in Example 11 have been quantitated, the issue of relative immunoreactivity of individual regions of the toxin A protein was precisely addressed.

For the purposes of this Example, the toxin A protein was subdivided into 6 intervals (1-6), numbered from the amino (interval 1) to the carboxyl (interval 6) termini.

The recombinant proteins corresponding to these intervals were from expression clones (see Example 11(d) for clone designations) pMA30-300 (interval 1), pMA300-660 (interval 2), pMA660-1100 (interval 3), pPA1100-1450 (interval 4), pMA1450-1870 (interval 5) mad pMA1870-2680 (interval 6). These 6 clones were selected because they span the entire protein from amino acids numbered 30 through 2680, and subdivide the protein into 6 small intervals. Also, the carbohydrate binding repeat interval is contained specifically in one interval (interval 6), allowing evaluation of the immune response specifically directed against this region. Western blots of 7.5% SDS-PAGE gels, loaded and electrophoresed with defined quantities of each recombinant protein, were probed with either goat antitoxin A polyclonal antibody (Tech Lab) or chicken antitoxin A polyclonal antibody pCTA IgY, Example 8(c)!. The blots were prepared and developed with alkaline phosphatase as previously described Williams et al. (1994), supra!. At least 90% of all reactivity, in either goat or chicken antibody pools, was found to be directed against the ligand binding domain (interval 6). The remaining immunoreactivity was directed against all five remaining intervals, and was similar in both antibody pools, except that the chicken antibody showed a much lower reactivity against interval 2 than the goat antibody.

This clearly demonstrates that when native toxin A is used as an immunogen in goats or chickens, the bulk of the immune response is directed against the ligand binding domain of the protein, with the remaining response distributed throughout the remaining 2/3 of the protein.

b) Affinity Purification Of IgY Reactive Against Recombinant Toxin A Protein

Affinity columns, containing recombinant toxin A protein from the 6 defined intervals in (a) above, were made and used to (i) affinity purify antibodies reactive to each individual interval from the CTA IgY preparation Example 8(c)!, and (ii) deplete interval specific antibodies from the CTA IgY preparation. Affinity columns were made by coupling 1 ml of PBS-washed Actigel resin (Sterogene) with region specific protein and 1/10 final volume of Ald-coupling solution (1M sodium cyanoborohydride). The total region specific protein added to each reaction mixture was 2.7 mg (interval 1), 3 mg (intervals 2 and 3), 0.1 mg (interval 4), 0.2 mg (interval 5) and 4 mg (interval 6). Protein for intervals 1, 3, and 6 was affinity purified pMAl fusion protein in column buffer (see Example 11). Interval 4 was affinity purified poly-histidine containing pET fusion in PBS; intervals 2 and 5 were from inclusion body preparations of insoluble pMAL fusion protein, dialyzed extensively in PBS. Aliquots of the supernatants from the coupling reactions, before and after coupling, were assessed by Coomassie staining of 7.5% SDS-PAGE gels. Based on protein band intensities, in all cases greater than 50% coupling efficiencies were estimated. The resins were poured into 5 ml BioRad columns, washed extensively with PBS, and stored at 4° C.

Aliquots of the CTA IgY polyclonal antibody preparation were depleted for each individual region as described below. A 20 ml sample of the CTA IgY preparation Example 8(c)! was dialyzed extensively against 3 changes of PBS (1 liter for each dialysis), quantitated by absorbance at OD₂₈₀, and stored at 4° C. Six 1 ml aliquots of the dialyzed IgY preparation were removed, and depleted individually for each of the six intervals. Each 1 ml aliquot was passed over the appropriate affinity column, and the eluate twice reapplied to the column. The eluate was collected, and pooled with a 1 ml PBS wash. Bound antibody was eluted from the column by washing with 5 column volumes of 4M Guanidine-HCl (in 10 mM Tris-HCl, pH 8.0). The column was reequilibrated in PBS, and the depleted antibody stock reapplied as described above. The eluate was collected, pooled with a 1 ml PBS wash, quantitated by absorbance at OD₂₈₀, and stored at 4° C. In this manner, 6 aliquots of the CTA IgY preparation were individually depleted for each of the 6 toxin A intervals, by two rounds of affinity depletion. The specificity of each depleted stock was tested by Western blot analysis. Multiple 7.5% SDS-PAGE gels were loaded with protein samples corresponding to all 6 toxin A subregions. After electrophoresis, the gels were blotted, and protein transfer confirmed by Ponceau S staining protocols described in Williams et al. (1994), supra!. After blocking the blots 1 hr at 20° C. in PBS+0.1% Tween 20 (PBST) containing 5% milk (as a blocking buffer), 4 ml of either a 1/500 dilution of the dialyzed CTA IgY preparation in blocking buffer, or an equivalent amount of the six depleted antibody stocks (using OD₂₈₀ to standardize antibody concentration) were added and the blots incubated a further 1 hr at room temperature. The blots were washed and developed with alkaline phosphatase (using a rabbit anti-chicken alkaline phosphate conjugate as a secondary antibody) as previously described Williams et al. (1994), supra!. In all cases, only the target interval was depleted for antibody reactivity, and at least 90% of the reactivity to the target intervals was specifically depleted.

Region specific antibody pools were isolated by affinity chromatography as described below. Ten mls of the dialyzed CTA IgY preparation were applied sequentially to each affinity column, such that a single 10 ml aliquot was used to isolate region specific antibodies specific to each of the six subregions. The columns were sequentially washed with 10 volumes of PBS, 6 volumes of BBS-Tween, 10 volumes of TBS, and eluted with 4 ml Actisep elution media (Sterogene). The eluate was dialyzed extensively against several changes of PBS, and the affinity purified antibody collected and stored at 4° C. The volumes of the eluate increased to greater than 10 mls during dialysis in each case, due to the high viscosity of the Actisep elution media. Aliquots of each sample were 20× concentrated using Centricon 30 microconcentrators (Amicon) and stored at 4° C. The specificity of each region specific antibody pool was tested, relative to the dialyzed CTA IgY preparation, by Western blot analysis, exactly as described above, except that 4 ml samples of blocking buffer containing 100 μl region specific antibody (unconcentrated) were used instead of the depleted CTA IgY preparations. Each affinity purified antibody preparation was specific to the defined interval, except that samples purified against intervals 1-5 also reacted with interval 6. This may be due to non-specific binding to the interval 6 protein, since this protein contains the repetitive ligand binding domain which has been shown to bind antibodies nonspecifically. Lyerly et al., Curr. Microbiol., 19:303-306 (1989).!

The reactivity of each affinity purified antibody preparation to the corresponding proteins was approximately the same as the reactivity of the 1/500 diluted dialyzed CTA IgY preparation standard. Given that the specific antibody stocks were diluted 1/40, this would indicate that the unconcentrated affinity purified antibody stocks contain 1/10-1/20 the concentration of specific antibodies relative to the starting CTA IgY preparation.

c) Toxin A Neutralization Assay Using Antibodies Reactive Toward Recombinant Toxin A Protein

The CHO toxin neutralization assay Example 8(d)! was used to assess the ability of the depleted or enriched samples generated in (b) above to neutralize the cytotoxicity of toxin A. The general ability of affinity purified antibodies to neutralize toxin A was assessed by mixing together aliquots of all 6 concentrated stocks of the 6 affinity purified samples generated in (b) above, and testing the ability of this mixture to neutralize a toxin A concentration of 0.1 μg/ml. The results, shown in FIG. 11, demonstrate almost complete neutralization of toxin A using the affinity purified (AP) mix. Some epitopes within the recombinant proteins utilized for affinity purification were probably lost when the proteins were denatured before affinity purification by Guanidine-HCl treatment in (b) above!. Thus, the neutralization ability of antibodies directed against recombinant protein is probably underestimated using these affinity purified antibody pools. This experiment demonstrates that antibodies reactive to recombinant toxin A can neutralize cytotoxicity, suggesting that neutralizing antibodies may be generated by using recombinant toxin A protein as immunogen.

In view of the observation that the recombinant expression clones of the toxin A gene divide the protein into 6 subregions, the neutralizing ability of antibodies directed against each individual region was assessed. The neutralizing ability of antibodies directed against the ligand binding domain of toxin A was determined first.

In the toxin neutralization experiment shown in FIG. 11, interval 6 specific antibodies (interval 6 contains the ligand binding domain) were depleted from the dialyzed PEG preparation, and the effect on toxin neutralization assayed. Interval 6 antibodies were depleted either by utilizing the interval 6 depleted CTA IgY preparation from (b) above ("-6 aff. depleted" in FIG. 11), or by addition of interval 6 protein to the CTA IgY preparation (estimated to be a 10 fold molar excess over anti-interval 6 immunoglobulin present in this preparation) to competitively compete for interval 6 protein ("-6 prot depleted" in FIG. 11). In both instances, removal of interval 6 specific antibodies reduces the neutralization efficiency relative to the starting CTA IgY preparation. This demonstrates that antibodies directed against interval 6 contribute to toxin neutralization. Since interval 6 corresponds to the ligand binding domain of the protein, these results demonstrate that antibodies directed against this region in the PEG preparation contribute to the neutralization of toxin A in this assay. However, it is significant that after removal of these antibodies, the PEG preparation retains significant ability to neutralize toxin A (FIG. 11). This neutralization is probably due to the action of antibodies specific to other regions of the toxin A protein, since at least 90% of the ligand binding region reactive antibodies were removed in the depleted sample prepared in (b) above. This conclusion was supported by comparison of the toxin neutralization of the affinity purified (AP) mix compared to affinity purified interval 6 antibody alone. Although some neutralization ability was observed with AP interval 6 antibodies alone, the neutralization was significantly less than that observed with the mixture of all 6 AP antibody stocks (not shown).

Given that the mix of all six affinity purified samples almost completely neutralized the cytotoxicity of toxin A (FIG. 11), the relative importance of antibodies directed against toxin A intervals 1-5 within the mixture was determined. This was assessed in two ways. First, samples containing affinity purified antibodies representing 5 of the 6 intervals were prepared, such that each individual region was depleted from one sample. FIG. 12 demonstrates a sample neutralization curve, comparing the neutralization ability of affinity purified antibody mixes without interval 4 (-4) or 5 (-5) specific antibodies, relative to the mix of all 6 affinity purified antibody stocks (positive control). While the removal of interval 5 specific antibodies had no effect on toxin neutralization (or intervals 1-3, not shown), the loss of interval 4 specific antibodies significantly reduced toxin neutralization (FIG. 12).

Similar results were seen in a second experiment, in which affinity purified antibodies, directed against a single region, were added to interval 6 specific antibodies, and the effects on toxin neutralization assessed. Only interval 4 specific antibodies significantly enhanced neutralization when added to interval 6 specific antibodies (FIG. 13). These results demonstrate that antibodies directed against interval 4 (corresponding to clone pPA1100-1450 in FIG. 9) are important for neutralization of cytotoxicity in this assay. Epitope mapping has shown that only low levels of antibodies reactive to this region are generated when native toxin A is used as an immunogen Example 12(a)!. It is hypothesized that immunization with recombinant protein specific to this interval will elicit higher titers of neutralizing antibodies. In summary, this analysis has identified two critical regions of the toxin A protein against which neutralizing antibodies are produced, as assayed by the CHO neutralization assay.

EXAMPLE 13 Production And Evaluation Of Avian Antitoxin Against C. difficile Recombinant Toxin A Polypeptide

In Example 12, we demonstrated neutralization of toxin A mediated cytotoxicity by affinity purified antibodies reactive to recombinant toxin A protein. To determine whether antibodies raised against a recombinant polypeptide fragment of C. difficile toxin A may be effective in treating clostridial diseases, antibodies to recombinant toxin A protein representing the binding domain were generated. Two toxin A binding domain recombinant polypeptides, expressing the binding domain in either the pMALc (pMA1870-2680) or pET 23(pPA1870-2680) vector, were used as immunogens. The pMAL protein was affinity purified as a soluble product Example 12(d)! and the pET protein was isolated as insoluble inclusion bodies Example 12(d)! and solubilized to an immunologically active protein using a proprietary method described in a pending patent application (U.S. patent application Ser. No. 08/129,027). This Example involves (a) immunization, (b) antitoxin collection, (c) determination of antitoxin antibody titer, (d) anti-recombinant toxin A neutralization of toxin A hemagglutination activity in vitro, and (e) assay of in vitro toxin A neutralizing activity.

a) Immunization

The soluble and the inclusion body preparations each were used separately to immunize hens. Both purified toxin A polypeptides were diluted in PBS and emulsified with approximately equal volumes of CFA for the initial immunization or IFA for subsequent booster immunizations. On day zero, for each of the recombinant preparations, two egg laying white Leghorn hens (obtained from local breeder) were each injected at multiple sites (intramuscular and subcutaneous) with 1 ml of recombinant adjuvant mixture containing approximately 0.5 to 1.5 mgs of recombinant toxin A. Booster immunizations of 1.0 mg were given on days 14 and day 28.

b) Antitoxin Collection

Total yolk immune IgY was extracted as described in the standard PEG protocol (as in Example 1) and the final IgY pellet was dissolved in sterile PBS at the original yolk volume. This material is designated "immune recombinant IgY" or "immune IgY."

c) Antitoxin Antibody Titer

To determine if the recombinant toxin A protein was sufficiently immunogenic to raise antibodies in hens, the antibody titer of a recombinant toxin A polypeptide was determined by ELISA. Eggs from both hens were collected on day 32, the yolks pooled and the antibody was isolated using PEG as described. The immune recombinant IgY antibody titer was determined for the soluble recombinant protein containing the maltose binding protein fusion generated in p-Mal (pMA1870-2680). Ninety-six well Falcon Pro-bind plates were coated overnight at 4° C. with 100 μl/well of toxin A recombinant at 2.5 μg/μl in PBS containing 0.05% thimerosal. Another plate was also coated with maltose binding protein (MBP) at the same concentration, to permit comparison of antibody reactivity to the fusion partner. The next day, the wells were blocked with PBS containing 1% bovine serum albumin (BSA) for 1 hour at 37° C. IgY isolated from immune or preimmune eggs was diluted in antibody diluent (PBS containing 1% BSA and 0.05% Tween-20), and added to the blocked wells and incubated for 1 hour at 37° C. The plates were washed three times with PBS with 0.05% Tween-20, then three times with PBS. Alkaline phosphatase conjugated rabbit anti-chicken IgG (Sigma) diluted 1:1000 in antibody diluent was added to the plate, and incubated for 1 hour at 37° C. The plates were washed as before and substrate was added, p-nitrophenyl phosphate (Sigma)! at 1 mg/ml in 0.05M Na₂ CO₃, pH 9.5 and 10 mM MgCl₂. The plates were evaluated quantitatively on a Dynatech MR 300 Micro EPA plate reader at 410 nm about 10 minutes after the addition of substrate.

Based on these ELISA results, high antibody titers were raised in chickens immunized with the toxin A recombinant polypeptide. The recombinant appeared to be highly immunogenic, as it was able to generate high antibody titers relatively quickly with few immunizations. Immune IgY titer directed specifically to the toxin A portion of the recombinant was higher than the immune IgY titer to its fusion partner, the maltose binding protein, and significantly higher than the preimmune IgY. ELISA titers (reciprocal of the highest dilution of IgY generating a signal) in the preimmune IgY to the MBP or the recombinant was <1:30 while the immune IgY titers to MBP and the toxin A recombinant were 1:18750 and >1:93750 respectively. Importantly, the anti-recombinant antibody titers generated in the hens against the recombinant polypeptide is much higher, compared to antibodies to that region raised using native toxin A. The recombinant antibody titer to region 1870-2680 in the CTA antibody preparation is at least five-fold lower compared to the recombinant generated antibodies (1:18750 versus >1:93750). Thus, it appears a better immune response can be generated against a specific recombinant using that recombinant as the immunogen compared to the native toxin A.

This observation is significant, as it shows that because recombinant portions stimulate the production of antibodies, it is not necessary to use native toxin molecules to produce antitoxin preparations. Thus, the problems associated with the toxicity of the native toxin are avoided and large-scale antitoxin production is facilitated.

d) Anti-Recombinant Toxin A Neutralization Of Toxin A Hemagglutination Activity In Vitro

Toxin A has hemagglutinating activity besides cytotoxic and enterotoxin properties. Specifically, toxin A agglutinates rabbit erythrocytes by binding to a trisaccharide (gal 1-3B1-4GlcNAc) on the cell surface. H. Krivan et al., Infect. Immun., 53:573-581 (1986).! We examined whether the anti-recombinant toxin A (immune IgY, antibodies raised against the insoluble product expressed in pET) can neutralize the hemagglutination activity of toxin A in vitro. The hemagglutination assay procedure used was described by H. C. Krivan et al. Polyethylene glycol-fractionated immune or preimmune IgY were pre-absorbed with citrated rabbit erythrocytes prior to performing the hemagglutination assay because we have found that IgY alone can agglutinate red blood cells. Citrated rabbit red blood cells (RRBC's)(Cocalico) were washed twice by centrifugation at 450×g with isotonic buffer (0.1 M Tris-HCl, 0.05M NaCl, pH 7.2). RRBC-reactive antibodies in the IgY were removed by preparing a 10% RRBC suspension (made by adding packed cells to immune or preimmune IgY) and incubating the mixture for 1 hour at 37° C. The RRBCs were then removed by centrifugation. Neutralization of the hemagglutination activity of toxin A by antibody was tested in round-bottomed 96-well microtiter plates. Twenty-five μl of toxin A (36 μg/ml) (Tech Lab) in isotonic buffer was mixed with an equal volume of different dilutions of immune or preimmune IgY in isotonic buffer, and incubated for 15 minutes at room temperature. Then, 50 μl of a 1% RRBC suspension in isotonic buffer was added and the mixture was incubated for 3 hours at 4° C. Positive control wells containing the final concentration of 9 μg/ml of toxin A after dilution without IgY were also included. Hemagglutination activity was assessed visually, with a diffuse matrix of RRBC's coating the bottom of the well representing a positive hemagglutination reaction and a tight button of RRBC's at the bottom of the well representing a negative reaction. The anti-recombinant immune IgY neutralized toxin A hemagglutination activity, giving a neutralization titer of 1:8. However, preimmune IgY was unable to neutralize the hemagglutination ability of toxin A.

e) Assay Of In Vitro Toxin A Neutralizing Activity

The ability of the anti-recombinant toxin A IgY (immune IgY antibodies raised against pMA1870-2680, the soluble recombinant binding domain protein expressed in pMAL, designated as Anti-tox. A-2 in FIG. 14, and referred to as recombinant region 6) and pre-immune IgY, prepared as described in Example 8(c) above, to neutralize the cytotoxic activity of toxin A was assessed in vitro using the CHO cell cytotoxicity assay, and toxin A (Tech Lab) at a concentration of 0.1 μg/ml, as described in Example 8(d) above. As additional controls, the anti-native toxin A IgY (CTA) and pre-immune IgY preparations described in Example 8(c) above were also tested. The results are shown in FIG. 14.

The anti-recombinant toxin A IgY demonstrated only partial neutralization of the cytotoxic activity of toxin A, while the pre-immune IgY did not demonstrate any significant neutralizing activity.

EXAMPLE 14 In vivo Neutralization Of C. difficile Toxin A

The ability of avian antibodies (IgY) raised against recombinant toxin A binding domain to neutralize the enterotoxin activity of C. difficile toxin A was evaluated in vivo using Golden Syrian hamsters. The Example involved: (a) preparation of the avian anti-recombinant toxin A IgY for oral administration; (b) in vivo protection of hamsters from C. difficile toxin A enterotoxicity by treatment of toxin A with avian anti-recombinant toxin A IgY; and (c) histologic evaluation of hamster ceca.

a) Preparation Of The Avian Anti-Recombinant Toxin A IgY For Oral Administration

Eggs were collected from hens which had been immunized with the recombinant C. difficile toxin A fragment pMA1870-2680 (described in Example 13, above). A second group of eggs purchased at a local supermarket was used as a pre-immune (negative) control. Egg yolk immunoglobulin (IgY) was extracted by PEG from the two groups of eggs as described in Example 8(c), and the final IgY pellets were solubilized in one-fourth the original yolk volume using 0.1M carbonate buffer (mixture of NaHCO₃ and Na₂ CO₃), pH 9.5. The basic carbonate buffer was used in order to protect the toxin A from the acidic pH of the stomach environment.

b) In vivo Protection Of Hamsters Against C. difficile Toxin A Enterotoxicity By Treatment Of Toxin A With Avian Anti-recombinant Toxin A IgY

In order to assess the ability of the arian anti-recombinant toxin A IgY, prepared in section (a) above to neutralize the in vivo enterotoxin activity of toxin A, an in vivo toxin neutralization model was developed using Golden Syrian hamsters. This model was based on published values for the minimum amount of toxin A required to elicit diarrhea (0.08 mg toxin A/Kg body wt.) and death (0.16 mg toxin A/Kg body wt.) in hamsters when administered orally (Lyerly et al. Infect. Immun., 47:349-352 (1985).

For the study, four separate experimental groups were used, with each group consisting of 7 female Golden Syrian hamsters (Charles River), approx. three and one-half weeks old, weighing approx. 50 gms each. The animals were housed as groups of 3 and 4, and were offered food and water ad libitum through the entire length of the study.

For each animal, a mixture containing either 10 μg of toxin A (0.2 mg/Kg) or 30 μg of toxin A (0.6 mg/Kg) (C. difficile toxin A was obtained from Tech Lab and 1 ml of either the anti-recombinant toxin A IgY or pre-immune IgY (from section (a) above) was prepared. These mixtures were incubated at 37° C. for 60 min. and were then administered to the animals by the oral route. The animals were then observed for the onset of diarrhea and death for a period of 24 hrs. following the administration of the toxin A+IgY mixtures, at the end of which time, the following results were tabulated and shown in Table 17:

                  TABLE 17                                                         ______________________________________                                         Study Outcome At 24 Hours                                                                       Study Outcome at 24 Hours                                     Experimental Group Healthy.sup.1                                                                           Diarrhea.sup.2                                                                          Dead.sup.3                                ______________________________________                                         10 μg Toxin A + Antitoxin Against                                                              7        0        0                                         Interval 6                                                                     30 μg Toxin A + Antitoxin Against                                                              7        0        0                                         Interval 6                                                                     10 μg Toxin A + Pre-Immune Serum                                                               0        5        2                                         30 μg Toxin A + Pre-Immune                                                                     0        5        2                                         ______________________________________                                          .sup.1 Animals remained healthy through the entire 24 hour study period.       .sup.2 Animals developed diarrhea, but did not die.                            .sup.3 Animals developed diarrhea, and subsequently died.                

Pretreatment of toxin A at both doses tested, using the anti-recombinant toxin A IgY, prevented all overt symptoms of disease in hamsters. Therefore, pretreatment of C. difficile toxin A, using the anti-recombinant toxin A IgY, neutralized the in vivo enterotoxin activity of the toxin A. In contrast, all animals from the two groups which received toxin A which had been pretreated using pre-immune IgY developed disease symptoms which ranged from diarrhea to death. The diarrhea which developed in the 5 animals which did not die in each of the two pre-immune groups, spontaneously resolved by the end of the 24 hr. study period.

c) Histologic Evaluation Of Hamster Ceca

In order to further assess the ability of anti-recombinant toxin A IgY to protect hamsters from the enterotoxin activity of toxin A, histologic evaluations were performed on the ceca of hamsters from the study described in section (b) above.

Three groups of animals were sacrificed in order to prepare histological specimens. The first group consisted of a single representative animal taken from each of the 4 groups of surviving hamsters at the conclusion of the study described in section (b) above. These animals represented the 24 hr. timepoint of the study.

The second group consisted of two animals which were not part of the study described above, but were separately treated with the same toxin A+pre-immune IgY mixtures as described for the animals in section (b) above. Both of these hamsters developed diarrhea, and were sacrificed 8 hrs. after the time of administration of the toxin A+pre-immune IgY mixtures. At the time of sacrifice, both animals were presenting symptoms of diarrhea. These animals represented the acute phase of the study.

The final group consisted of a single untreated hamster from the same shipment of animals as those used for the two previous groups. This animal served as the normal control.

Samples of cecal tissue were removed from the 7 animals described above, and were fixed overnight at 4° C. using 10% buffered formalin. The fixed tissues were paraffin-embedded, sectioned, and mounted on glass microscope slides. The tissue sections were then stained using hematoxylin and eosin (H and E stain), and were examined by light microscopy.

The tissues obtained from the two 24 hr. animals which received mixtures containing either 10 μg or 30 μg of toxin A and anti-recombinant toxin A IgY were indistinguishable from the normal control, both in terms of gross pathology, as well as at the microscopic level. These observations provide further evidence for the ability of anti-recombinant toxin A IgY to effectively neutralize the in vivo enterotoxin activity of C. difficile toxin A, and thus its ability to prevent acute or lasting toxin A-induced pathology.

In contrast, the tissues from the two 24 hr. animals which received the toxin A+pre-immune IgY mixtures demonstrated significant pathology. In both of these groups, the mucosal layer was observed to be less organized than in the normal control tissue. The cytoplasm of the epithelial cells had a vacuolated appearance, and gaps were present between the epithelium and the underlying cell layers. The lamina propria was largely absent. Intestinal villi and crypts were significantly diminished, and appeared to have been overgrown by a planar layer of epithelial cells and fibroblasts. Therefore, although these animals overtly appeared to recover from the acute symptoms of toxin A intoxication, lasting pathologic alterations to the cecal mucosa had occurred.

The tissues obtained from the two acute animals which received mixtures of toxin A and pre-immune IgY demonstrated the most significant pathology. At the gross pathological level, both animals were observed to have severely distended ceca which were filled with watery, diarrhea-like material. At the microscopic level, the animal that was given the mixture containing 10 μg of toxin A and pre-immune IgY was found to have a mucosal layer which had a ragged, damaged appearance, and a disorganized, compacted quality. The crypts were largely absent, and numerous breaks in the epithelium had occurred. There was also an influx of erythrocytes into spaces between the epithelial layer and the underlying tissue. The animal which had received the mixture containing 30 μg of toxin A and pre-immune IgY demonstrated the most severe pathology. The cecal tissue of this animal had an appearance very similar to that observed in animals which had died from C. difficile disease. Widespread destruction of the mucosa was noted, and the epithelial layer had sloughed. Hemorrhagic areas containing large numbers of erythrocytes were very prevalent. All semblance of normal tissue architecture was absent from this specimen. In terms of the presentation of pathologic events, this in vivo hamster model of toxin A-intoxication correlates very closely with the pathologic consequences of C. difficile disease in hamsters. The results presented in this Example demonstrate that while anti-recombinant toxin A (Interval 6) IgY is capable of only partially neutralizing the cytotoxic activity of C. difficile toxin A, the same antibody effectively neutralizes 100% of the in vivo enterotoxin activity of the toxin. While it is not intended that this invention be limited to this mechanism, this may be due to the cytotoxicity and enterotoxicity of C. difficile Toxin A as two separate and distinct biological functions.

EXAMPLE 15 In Vivo Neutralization Of C. difficile Toxin A By Antibodies Against Recombinant Toxin A Polypeptides

The ability of avian antibodies directed against the recombinant C. difficile toxin A fragment 1870-2680 (as expressed by pMA1870-2680; see Example 13) to neutralize the enterotoxic activity of toxin A was demonstrated in Example 14. The ability of avian antibodies (IgYs) directed against other recombinant toxin A epitopes to neutralize native toxin A in vivo was next evaluated. This example involved: (a) the preparation of IgYs against recombinant toxin A polypeptides; (b) in vivo protection of hamsters against toxin A by treatment with anti-recombinant toxin A IgYs and (c) quantification of specific antibody concentration in CTA and Interval 6 IgY PEG preparations.

The nucleotide sequence of the coding region of the entire toxin A protein is listed in SEQ ID NO:5. The amino acid sequence of the entire toxin A protein is listed in SEQ ID NO:6. The amino acid sequence consisting of amino acid residues 1870 through 2680 of toxin A is listed in SEQ ID NO:7. The amino acid sequence consisting of amino acid residues 1870 through 1960 of toxin A is listed in SEQ ID NO:8. The amino acid sequence of residues 1873 through 2684 of toxin A is listed in SEQ ID NO:29.

a) Preparation Of IgY's Against Recombinant Toxin A Polypeptides

Eggs were collected from Leghorn hens which have been immunized with recombinant C. difficile toxin A polypeptide fragments encompassing the entire toxin A protein. The polypeptide fragments used as immunogens were: 1) pMA 1870-2680 (Interval 6), 2) pPA 1100-1450 (Interval 4), and 3) a mixture of fragments consisting of pMA 30-300 (Interval 1), pMA 300-660 (Interval 2), pMA 660-1100 (Interval 3) and pMA 1450-1870 (Interval 5). This mixture of immunogens is referred to as Interval 1235. The location of each interval within the toxin A molecule is shown in FIG. 15A. In FIG. 15A, the following abbreviations are used: pP refers to the pET23 vector (New England BioLabs); pM refers to the pMAL™-c vector (New England BioLabs); A refers to toxin A; the numbers refer to the amino acid interval expressed in the clone. (For example, the designation pMA30-300 indicates that the recombinant clone encodes amino acids 30-300 of toxin A and the vector used was pMAL™-c).

The recombinant proteins were generated as described in Example 11. The IgYs were extracted and solubilized in 0.1M carbonate buffer pH 9.5 for oral administration as described in Example 14(a). The IgY reactivities against each individual recombinant interval was evaluated by ELISA as described in Example 13(c).

b) In Vivo Protection Of Hamsters Against Toxin A By Treatment With Anti-Recombinant Toxin A Antibodies

The ability of antibodies raised against recombinant toxin A polypeptides to provide in vivo protection against the enterotoxic activity of toxin A was examined in the hamster model system. This assay was performed as described in Example 14(b). Briefly, for each 40-50 gram female Golden Syrian hamster (Charles River), 1 ml of IgY 4X (i.e., resuspended in 1/4 of the original yolk volume) PEG prep against Interval 6, Interval 4 or Interval 1235 was mixed with 30 μg (LD₁₀₀ oral dose) of C. difficile toxin A (Tech Lab). Preimmune IgY mixed with toxin A served as a negative control. Antibodies raised against C. difficile toxoid A (Example 8) mixed with toxin A (CTA) served as a positive control. The mixture was incubated for 1 hour at 37° C. then orally administered to lightly etherized hamsters using an 18G feeding needle. The animals were then observed for the onset of diarrhea and death for a period of approximately 24 hours. The results are shown in Table 18.

                  TABLE 18                                                         ______________________________________                                         Study Outcome After 24 Hours                                                   Treatment group                                                                            Healthy.sup.1                                                                              Diarrhea.sup.2                                                                          Dead.sup.3                                    ______________________________________                                         Preimmune   0           0        7                                             CTA         5           0        0                                             Interval 6  6           1        0                                             Interval 4  0           1        6                                             Interval 1235                                                                              0           0        7                                             ______________________________________                                          .sup.1 Animal shows no sign of illness.                                        .sup.2 Animal developed diarrhea, but did not die.                             .sup.3 Animal developed diarrhea and died.                               

Pre-treatment of toxin A with IgYs against Interval 6 prevented diarrhea in 6 of 7 hamsters and completely prevented death in all 7. In contrast, as with preimmune IgY, IgYs against Interval 4 and Interval 1235 had no effect on the onset of diarrhea and death in the hamsters.

c) Quantification Of Specific Antibody Concentration In CTA And Interval 6 IgY PEG Preparations

To determine the purity of IgY PEG preparations, an aliquot of a pMA1870-2680 (Interval 6) IgY PEG preparation was chromatographed using HPLC and a KW-803 sizing column (Shodex). The resulting profile of absorbance at 280 nm is shown in FIG. 16. The single large peak corresponds to the predicted MW of IgY. Integration of the area under the single large peak showed that greater than 95% of the protein eluted from the column was present in this single peak. This result demonstrated that the majority (>95%) of the material absorbing at 280 nm in the PEG preparation corresponds to IgY. Therefore, absorbance at 280 nm can be used to determine the total antibody concentration in PEG preparations.

To determine the concentration of Interval 6-specific antibodies (expressed as percent of total antibody) within the CTA and pMA1870-2680 (Interval 6) PEG preparations, defined quantities of these antibody preparations were affinity purified on a pPA1870-2680(H) (shown schematically in FIG. 15B) affinity column and the specific antibodies were quantified. In FIG. 15B the following abbreviations are used: pP refers to the pET23 vector (New England BioLabs); pM refers to the pMAL™-c vector (New England BioLabs); pG refers to the pGEX vector (Pharmacia); pB refers to the PinPoint™ Xa vector (Promega); A refers to toxin A; the numbers refer to the amino acid interval expressed in the clone. The solid black ovals represent the MBP; the hatched ovals represent glutathione S-transferase; the hatched circles represent the biotin tag; and HHH represents the poly-histidine tag.

An affinity column containing recombinant toxin A repeat protein was made as follows. Four ml of PBS-washed Actigel resin (Sterogene) was coupled with 5-10 mg of pPA1870-2680 inclusion body protein prepared as described in Example (17) and dialyzed into PBS! in a 15 ml tube (Falcon) containing 1/10 final volume Ald-coupling solution (1M sodium cyanoborohydride). Aliquots of the supernatant from the coupling reactions, before and after coupling, were assessed by Coomassie staining of 7.5% SDS-PAGE gels. Based upon protein band intensities, greater than 6 mg of recombinant protein was coupled to the resin. The resin was poured into a 10 ml column (BioRad), washed extensively with PBS, pre-eluted with 4M guanidine-HCl (in 10 mM Tris-HCl, pH 8.0; 0.005% thimerosal) and re-equilibrated with PBS. The column was stored at 4° C.

Aliquots of a pMA1870-2680 (Interval 6) or a CTA IgY polyclonal antibody preparation (PEG prep) were affinity purified on the above affinity column as follows. The column was attached to an UV monitor (ISCO) and washed with PBS. For pMA1870-2680 IgY purification, a 2X PEG prep (filter sterilized using a 0.45μ filter; approximately 500 mg total IgY) was applied. The column was washed with PBS until the baseline was re-established (the column flow-through was saved), washed with BBSTween to elute nonspecifically binding antibodies and re-equilibrated with PBS. Bound antibody was eluted from the column in 4M guanidine-HCl (in 10 mM Tris-HCl, pH 8.0; 0.005% thimerosal). The entire elution peak was collected in a 15 ml tube (Falcon). The column was re-equilibrated and the column eluate was re-chromatographed as described above. The antibody preparation was quantified by UV absorbance (the elution buffer was used to zero the spectrophotometer). Total purified antibody was approximately 9 mg and 1 mg from the first and second chromatography passes, respectively. The low yield from the second pass indicated that most specific antibodies were removed by the first round of chromatography. The estimated percentage of Interval 6 specific antibodies in the pMA1870-2680 PEG prep is approximately 2%.

The percentage of Interval 6 specific antibodies in the CTA PEG prep was determined (utilizing the same column and methodology described above) to be approximately 0.5% of total IgY.

A 4X PEG prep contains approximately 20 mg/ml IgY. Thus in b) above, approximately 400 μg specific antibody in the Interval 6 PEG prep neutralized 30 μg toxin A in vivo.

EXAMPLE 16 In Vivo Treatment Of C. difficile Disease In Hamsters By Recombinant Interval 6 Antibodies

The ability of antibodies directed against recombinant Interval 6 of toxin A to protect hamsters in vivo from C. difficile disease was examined. This example involved: (a) prophylactic treatment of C. difficile disease and (b) therapeutic treatment of C. difficile disease.

a) Prophylactic Treatment Of C. difficile Disease

This experiment was performed as described in Example 9(b). Three groups each consisting of 7 female 100 gram Syrian hamsters (Charles River) were prophylactically treated with either preimmune IgYs, IgYs against native toxin A and B CTAB; see Example 8 (a) and (b)! or IgYs against Interval 6. IgYs were prepared as 4X PEG preparations as described in Example 9(a).

The animals were orally dosed 3 times daily, roughly at 4 hour intervals, for 12 days with 1 ml antibody preparations diluted in Ensure®. Using estimates of specific antibody concentration from Example 15(c), each dose of the Interval 6 antibody prep contained approximately 400 μg of specific antibody. On day 2 each hamster was predisposed to C. difficile infection by the oral administration of 3.0 mg of Clindamycin-HCl (Sigma) in 1 ml of water. On day 3 the hamsters were orally challenged with 1 ml of C. difficile inoculum strain ATCC 43596 in sterile saline containing approximately 100 organisms. The animals were then observed for the onset of diarrhea and subsequent death during the treatment period. The results are shown in Table 19.

                  TABLE 19                                                         ______________________________________                                         Lethality After 12 Days Of Treatment                                           Treatment Group                                                                           Number Animals Alive                                                                          Number Animals Dead                                  ______________________________________                                         Preimmune  0              7                                                    CTAB       6              1                                                    Interval 6 7              0                                                    ______________________________________                                    

Treatment of hamsters with orally-administered IgYs against Interval 6 successfully protected 7 out of 7 (100%) of the animals from C. difficile disease. One of the hamsters in this group presented with diarrhea which subsequently resolved during the course of treatment. As shown previously in Example 9, antibodies to native toxin A and toxin B were highly protective. In this Example, 6 out of 7 animals survived in the CTAB treatment group. All of the hamsters treated with preimmune sera came down with diarrhea and died. The survivors in both the CTAB and Interval 6 groups remained healthy throughout a 12 day post-treatment period. In particular, 6 out of 7 Interval 6-treated hamsters survived at least 2 weeks after termination of treatment which suggests that these antibodies provide a long-lasting cure. These results represent the first demonstration that antibodies generated against a recombinant region of toxin A can prevent CDAD when administered passively to animals. These results also indicate that antibodies raised against Interval 6 alone may be sufficient to protect animals from C. difficile disease when administered prophylactically.

Previously others had raised antibodies against toxin A by actively immunizing hamsters against a recombinant polypeptide located within the Interval 6 region Lyerly, D. M., et al. (1990) Curr. Microbiol. 21:29!. FIG. 17 shows schematically the location of the Lyerly, et al. intra-Interval 6 recombinant protein (cloned into the pUC vector) in comparison with the complete Interval 6 construct (pMA1870-2680) used herein to generate neutralizing antibodies directed against toxin A. In FIG. 17, the solid black oval represents the MBP which is fused to the toxin A Interval 6 in pMA1870-2680.

The Lyerly, et al. antibodies (intra-Interval 6) were only able to partially protect hamsters against C. difficile infection in terms of survival (4 out of 8 animals survived) and furthermore, these antibodies did not prevent diarrhea in any of the animals. Additionally, animals treated with the intra-Interval 6 antibodies Lyerly, et al. (1990), supra! died when treatment was removed.

In contrast, the experiment shown above demonstrates that passive administration of anti-Interval 6 antibodies prevented diarrhea in 6 out of 7 animals and completely prevented death due to CDAD. Furthermore, as discussed above, passive administration of the anti-Interval 6 antibodies provides a long lasting cure (i.e., treatment could be withdrawn without incident).

b) Therapeutic Treatment Of C. difficile Disease: In Vivo Treatment Of An Established C. difficile Infection In Hamsters With Recombinant Interval 6 Antibodies

The ability of antibodies against recombinant interval 6 of toxin A to therapeutically treat C. difficile disease was examined. The experiment was performed essentially as described in Example 10(b). Three groups, each containing seven to eight female Golden Syrian hamsters (100 g each; Charles River) were treated with either preimmune IgY, IgYs against native toxin A and toxin B (CTAB) and IgYs against Interval 6. The antibodies were prepared as described above as 4X PEG preparations.

The hamsters were first predisposed to C. difficile infection with a 3 mg dose of Clindamycin-HCl (Sigma) administered orally in 1 ml of water. Approximately 24 hrs later, the animals were orally challenged with 1 ml of C. difficile strain ATCC 43596 in sterile saline containing approximately 200 organisms. One day after infection, the presence of toxin A and B was determined in the feces of the hamsters using a commercial immunoassay kit (Cytoclone A+B EPA, Cambridge Biotech) to verify establishment of infection. Four members of each group were randomly selected and tested. Feces from an uninfected hamster was tested as a negative control. All infected animals tested positive for the presence of toxin according to the manufacturer's procedure. The initiation of treatment then started approximately 24 hr post-infection.

The animals were dosed daily at roughly 4 hr intervals with 1 ml antibody preparation diluted in Ensure® (Ross Labs). The amount of specific antibodies given per dose (determined by affinity purification) was estimated to be about 400 μg of anti-Interval 6 IgY (for animals in the Interval 6 group) and 100 μg and 70 μg of anti-toxin A (Interval 6-specific) and anti-toxin B (Interval 3-specific; see Example 19), respectively, for the CTAB preparation. The animals were treated for 9 days and then observed for an additional 4 days for the presence of diarrhea and death. The results indicating the number of survivors and the number of dead 4 days post-infection are shown in Table 20.

                  TABLE 20                                                         ______________________________________                                         In vivo Therapeutic Treatment With Interval 6 Antibodies                       Treatment Group                                                                           Number Animals Alive                                                                          Number Animals Dead                                  ______________________________________                                         Preimmune  4              3                                                    CTAB       8              0                                                    Interval 6 8              0                                                    ______________________________________                                    

Antibodies directed against both Interval 6 and CTAB successfully prevented death from C. difficile when therapeutically administered 24 hr after infection. This result is significant since many investigators begin therapeutic treatment of hamsters with existing drugs (e.g., vancomycin, phenelfamycins, tiacumicins, etc.) 8 hr post-infection Swanson, et al. (1991)Antimicrobial Agents and Chemotherapy 35:1108 and (1989) J. Antibiotics 42:94!.

Forty-two percent of hamsters treated with preimmune IgY died from CDAD. While the anti-Interval 6 antibodies prevented death in the treated hamsters, they did not eliminate all symptoms of CDAD as 3 animals presented with slight diarrhea. In addition, one CTAB-treated and one preimmune-treated animal also had diarrhea 14 days post-infection. These results indicate that anti-Interval 6 antibodies provide an effective means of therapy for CDAD.

EXAMPLE 17 Induction Of Toxin A Neutralizing Antibodies Requires Soluble Interval 6 Protein

As shown in Examples 11(d) and 15, expression of recombinant proteins in E. coli may result in the production of either soluble or insoluble protein. If insoluble protein is produced, the recombinant protein is solubilized prior to immunization of animals. To determine whether, one or both of the soluble or insoluble recombinant proteins could be used to generate neutralizing antibodies to toxin A, the following experiment was performed. This example involved a) expression of the toxin A repeats and subfragments of these repeats in E. coli using a variety of expression vectors; b) identification of recombinant toxin A repeats and sub-regions to which neutralizing antibodies bind; and c) determination of the neutralization ability of antibodies raised against soluble and insoluble toxin A repeat immunogen.

a) Expression Of The Toxin A Repeats And Subfragments Of These Repeats In E. coli Using A Variety Of Expression Vectors

The interval 6 immunogen utilized in Examples 15 and 16 was the pMA1870-2680 protein, in which the toxin A repeats are expressed as a soluble fusion protein with the MBP (described in Example 11). Interestingly, expression of this region (from the SpeI site to the end of the repeats, see FIG. 15B) in three other expression constructs, as either native (pPA1870-2680), poly-His tagged pPA1870-2680 (H)! or biotin-tagged (pBA1870-2680) proteins resulted in completely insoluble protein upon induction of the bacterial host (see FIG. 15B). The host strain BL21 (Novagen) was used for expression of pBA1870-2680 and host strain BL21(DE3) (Novagen) was used for expression of pPA1870-2680 and pPA1870-2680(H). These insoluble proteins accumulated to high levels in inclusion bodies. Expression of recombinant plasmids in E. coli host cells grown in 2X YT medium was performed as described Williams, et al. (1994), supra!.

As summarized in FIG. 15B, expression of fragments of the toxin A repeats (as either N-terminal SpeI-EcoRI fragments, or C-terminal EcoRI-end fragments) also yielded high levels of insoluble protein using pGEX (pGA1870-2190), PinPoint™-Xa (pBA1870-2190 and pBA2250-2680) and pET expression systems (pPA1870-2190). The pGEX and pET expression systems are described in Example 11. The PinPoint™-Xa expression system drives the expression of fusion proteins in E. coli. Fusion proteins from PinPoint™-Xa vectors contain a biotin tag at the amino-terminal end and can be affinity purified SoftLink™ Soft Release avidin resin (Promega) under mild denaturing conditions (5 mM biotin).

The solubility of expressed proteins from the pPG 1870-2190 and pPA 1870-2190 expression constructs was determined after induction of recombinant protein expression under conditions reported to enhance protein solubility These conditions comprise growth of the host at reduced temperature (30° C.) and the utilization of high (1 mM IPTG) or low (0.1 mM IPTG) concentrations of inducer Williams et al. (1994), supra!. All expressed recombinant toxin A protein was insoluble under these conditions. Thus, expression of these fragments of the toxin A repeats in pET and pGEX expression vectors results in the production of insoluble recombinant protein even when the host cells are grown at reduced temperature and using lower concentrations of the inducer. Although expression of these fragments in pMal vectors yielded affinity purifiable soluble fusion protein, the protein was either predominantly insoluble (pMA1870-2190) or unstable (pMA2250-2650). Attempts to solubilize expressed protein from the pMA1870-2190 expression construct using reduced temperature or lower inducer concentration (as described above) did not improve fusion protein solubility.

Collectively, these results demonstrate that expression of the toxin A repeat region in E. coli results in the production of insoluble recombinant protein, when expressed as either large (aa 1870-2680) or small (aa 1870-2190 or aa 2250-2680) fragments, in a variety of expression vectors (native or poly-his tagged pET, pGEX or PinPoint™-Xa vectors), utilizing growth conditions shown to enhance protein solubility. The exception to this rule were fusions with the MBP, which enhanced protein solubility, either partially (pMA1870-2190) or fully (pMA1870-2680).

b) Identification Of Recombinant Toxin A Repeats And Sub-Regions To Which Neutralizing Antibodies Bind

Toxin A repeat regions to which neutralizing antibodies bind were identified by utilizing recombinant toxin A repeat region proteins expressed as soluble or insoluble proteins to deplete protective antibodies from a polyclonal pool of antibodies against native C. difficile toxin A. An in vivo assay was developed to evaluate proteins for the ability to bind neutralizing antibodies.

The rational for this assay is as follows. Recombinant proteins were first pre-mixed with antibodies against native toxin A (CTA antibody; generated in Example 8) and allowed to react. Subsequently, C. difficile toxin A was added at a concentration lethal to hamsters and the mixture was administered to hamsters via IP injection. If the recombinant protein contains neutralizing epitopes, the CTA antibodies would lose their ability to bind toxin A resulting in diarrhea and/or death of the hamsters.

The assay was performed as follows. The lethal dose of toxin A when delivered orally to nine 40 to 50 g Golden Syrian hamsters (Sasco) was determined to be 10 to 30 μg. The PEG-purified CTA antibody preparation was diluted to 0.5X concentration (i.e., the antibodies were diluted at twice the original yolk volume) in 0.1M carbonate buffer, pH 9.5. The antibodies were diluted in carbonate buffer to protect them from acid degradation in the stomach. The concentration of 0.5X was used because it was found to be the lowest effective concentration against toxin A. The concentration of Interval 6-specific antibodies in the 0.5X CTA prep was estimated to be 10-15 μg/ml (estimated using the method described in Example 15).

The inclusion body preparation insoluble Interval 6 protein; pPA1870-2680(H)! and the soluble Interval 6 protein pMA1870-2680; see FIG. 15! were both compared for their ability to bind to neutralizing antibodies against C. difficile toxin A (CTA). Specifically, 1 to 2 mg of recombinant protein was mixed with 5 ml of a 0.5X CTA antibody prep (estimated to contain 60-70 μg of Interval 6-specific antibody). After incubation for 1 hr at 37° C., CTA (Tech Lab) at a final concentration of 30 μg/ml was added and incubated for another 1 hr at 37° C. One ml of this mixture containing 30 μg of toxin A (and 10-15 μg of Interval 6-specific antibody) was administered orally to 40-50 g Golden Syrian hamsters (Sasco). Recombinant proteins that result in the loss of neutralizing capacity of the CTA antibody would indicate that those proteins contain neutralizing epitopes. Preimmune and CTA antibodies (both at 0.5X) without the addition of any recombinant protein served as negative and positive controls, respectively.

Two other inclusion body preparations, both expressed as insoluble products in the pET vector, were tested; one containing a different insert (toxin B fragment) other than Interval 6 called pPB1850-2070 (see FIG. 18) which serves as a control for insoluble Interval 6, the other was a truncated version of the Interval 6 region called pPA1870-2190 (see FIG. 15B). The results of this experiment are shown in Table 21.

                  TABLE 21                                                         ______________________________________                                         Binding Of Neutralizing Antibodies By Soluble                                  Interval 6 Protein Study Outcome After 24 Hours                                Treatment Group.sup.1                                                                         Healthy.sup.2                                                                            Diarrhea.sup.3                                                                            Dead.sup.4                                 ______________________________________                                         Preimmune Ab   0         3          2                                          CTA Ab         4         1          0                                          CTA Ab + Int 6 (soluble)                                                                      1         2          2                                          CTA Ab + Int 6 (insoluble)                                                                    5         0          0                                          CTA Ab + pPB1850-2070                                                                         5         0          0                                          CTA Ab + pPA1870-2190                                                                         5         0          0                                          ______________________________________                                          .sup.1 C. difficile toxin A (CTA) was added to each group.                     .sup.2 Animals showed no signs of illness.                                     .sup.3 Animals developed diarrhea but did not die.                             .sup.4 Animals developed diarrhea and died.                              

Preimmune antibody was ineffective against toxin A, while anti-CTA antibodies at a dilute 0.5X concentration almost completely protected the hamsters against the enterotoxic effects of CTA. The addition of recombinant proteins pPB1850-2070 or pPA1870-2190 to the anti-CTA antibody had no effect upon its protective ability, indicating that these recombinant proteins do not bind to neutralizing antibodies. On the other hand, recombinant Interval 6 protein was able to bind to neutralizing anti-CTA antibodies and neutralized the in vivo protective effect of the anti-CTA antibodies. Four out of five animals in the group treated with anti-CTA antibodies mixed with soluble Interval 6 protein exhibited toxin associated toxicity (diarrhea and death). Moreover, the results showed that Interval 6 protein must be expressed as a soluble product in order for it to bind to neutralizing anti-CTA antibodies since the addition of insoluble Interval 6 protein had no effect on the neutralizing capacity of the CTA antibody prep.

c) Determination Of Neutralization Ability Of Antibodies Raised Against Soluble And Insoluble Toxin A Repeat Immunogen

To determine if neutralizing antibodies are induced against solubilized inclusion bodies, insoluble toxin A repeat protein was solubilized and specific antibodies were raised in chickens. Insoluble pPA1870-2680 protein was solubilized using the method described in Williams et al. (1994), supra. Briefly, induced cultures (500 ml) were pelleted by centrifugation at 3,000×g for 10 min at 4° C. The cell pellets were resuspended thoroughly in 10 ml of inclusion body sonication buffer (25 mM HEPES pH 7.7, 100 mM KCl, 12.5 mM MgCl₂, 20% glycerol, 0.1% (v/v) Nonidet P-40, 1 mM DTT). The suspension was transferred to a 30 ml non-glass centrifuge tube. Five hundred μl of 10 mg/ml lysozyme was added and the tubes were incubated on ice for 30 min. The suspension was then frozen at -70° C. for at least 1 hr. The suspension was thawed rapidly in a water bath at room temperature and then placed on ice. The suspension was then sonicated using at least eight 15 sec bursts of the microprobe (Branson Sonicator Model No. 450) with intermittent cooling on ice.

The sonicated suspension was transferred to a 35 ml Oakridge tube and centrifuged at 6,000×g for 10 min at 4° C. to pellet the inclusion bodies. The pellet was washed 2 times by pipetting or vortexing in fresh, ice-cold RIPA buffer 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate in TBS (25 mM Tris-Cl pH 7.5, 150 mM NaCl)!. The inclusion bodies were recentrifuged after each wash. The inclusion bodies were dried and transferred using a small metal spatula to a 15 ml tube (Falcon). One ml of 10% SDS was added and the pellet was solubilized by gently pipetting the solution up and down using a 1 ml micropipettor. The solubilization was facilitated by heating the sample to 95° C. when necessary.

Once the inclusion bodies were in solution, the samples were diluted with 9 volumes of PBS. The protein solutions were dialyzed overnight against a 100-fold volume of PBS containing 0.05% SDS at room temperature. The dialysis buffer was then changed to PBS containing 0.01% SDS and the samples were dialyzed for several hours to overnight at room temperature. The samples were stored at 4° C. until used. Prior to further use, the samples were warmed to room temperature to allow any precipitated SDS to go back into solution.

The inclusion body preparation was used to immunize hens. The protein was dialyzed into PBS and emulsified with approximately equal volumes of CFA for the initial immunization or IFA for subsequent booster immunizations. On day zero, for each of the recombinant recombinant preparations, two egg laying white Leghorn hens were each injected at multiple sites (IM and SC) with 1 ml of recombinant protein-adjuvant mixture containing approximately 0.5-1.5 mg of recombinant protein. Booster immunizations of 1.0 mg were given of days 14 and day 28. Eggs were collected on day 32 and the antibody isolated using PEG as described in Example 14(a). High titers of toxin A specific antibodies were present (as assayed by ELISA, using the method described in Example 13). Titers were determined for both antibodies against recombinant polypeptides pPA1870-2680 and pMA1870-2680 and were found to be comparable at >1:62,500.

Antibodies against soluble Interval 6 (pMA1870-2680) and insoluble Interval 6 (inclusion body), pPA1870-2680! were tested for neutralizing ability against toxin A using the in vivo assay described in Example 15(b). Preimmune antibodies and antibodies against toxin A (CTA) served as negative and positive controls, respectively. The results are shown in Table 22.

                  TABLE 22                                                         ______________________________________                                         Neutralization Of Toxin A By Antibodies Against                                Soluble Interval 6 Protein Study Outcome After 24 Hours                        Antibody                                                                       Treatment Group                                                                               Healthy.sup.1                                                                            Diarrhea.sup.2                                                                            Dead.sup.3                                 ______________________________________                                         Preimmune      1         0          4                                          CTA            5         0          0                                          Interval 6 (Soluble).sup.4                                                                    5         0          0                                          Interval 6 (Insoluble)                                                                        0         2          3                                          ______________________________________                                          .sup.1 Animals showed no sign of illness.                                      .sup.2 Animal developed diarrhea but did not die.                              .sup.3 Animal developed diarrhea and died.                                     .sup.4 400 μg/ml                                                      

Antibodies raised against native toxin A were protective while preimmune antibodies had little effect. As found using the in vitro CHO assay described in Example 8(d)! where antibodies raised against the soluble Interval 6 could partially neutralize the effects of toxin A, here they were able to completely neutralize toxin A in vivo. In contrast, the antibodies raised against the insoluble Interval 6 was unable to neutralize the effects of toxin A in vivo as shown above (Table 22) and in vitro as shown in the CHO assay described in Example 8(d)!.

These results demonstrate that soluble toxin A repeat immunogen is necessary to induce the production of neutralizing antibodies in chickens, and that the generation of such soluble immunogen is obtained only with a specific expression vector (pMal) containing the toxin A region spanning aa 1870-2680. That is to say, insoluble protein that is subsequently solubilized does not result in a toxin A antigen that will elicit a neutralizing antibody.

EXAMPLE 18 Cloning And Expression Of The C. difficile Toxin B Gene

The toxin B gene has been cloned and sequenced; the amino acid sequence deduced from the cloned nucleotide sequence predicts a MW of 269.7 kD for toxin B Barroso et al., Nucl. Acids Res. 18:4004 (1990)!. The nucleotide sequence of the coding region of the entire toxin B gene is listed in SEQ ID NO:9. The amino acid sequence of the entire toxin B protein is listed in SEQ ID NO:10.

Given the expense and difficulty of isolating native toxin B protein, it would be advantageous to use simple and inexpensive procaryotic expression systems to produce and purify high levels of recombinant toxin B protein for immunization purposes. Ideally, the isolated recombinant protein would be soluble in order to preserve native antigenicity, since solubilized inclusion body proteins often do not fold into native conformations. Indeed as shown in Example 17, neutralizing antibodies against recombinant toxin A were only obtained when soluble recombinant toxin A polypeptides were used as the immunogen. To allow ease of purification, the recombinant protein should be expressed to levels greater than 1 mg/liter of E. coli culture.

To determine whether high levels of recombinant toxin B protein could be produced in E. coli, fragments of the toxin B gene were cloned into various prokaryotic expression vectors, and assessed for the ability to express recombinant toxin B protein in E. coli. This Example involved (a) cloning of the toxin B gene and (b) expression of the toxin B gene in E. coli.

a) Cloning Of The Toxin B Gene

The toxin B gene was cloned using PCR amplification from C. difficile genomic DNA. Initially, the gene was cloned in two overlapping fragments, using primer pairs P5/P6 and P7/P8. The location of these primers along the toxin B gene is shown schematically in FIG. 18. The sequence of each of these primers is:

P5: 5' TAGAAAAAATGGCAAATGT 3' (SEQ ID NO:11);

P6: 5' TTTCATCTTGTA GAGTCAAAG 3' (SEQ ID NO:12);

P7: 5' GATGCCACAAGATGATTTAGTG 3' (SEQ ID NO:13); and

P8: 5' CTAATTGAGCTGTATCAGGATC 3' (SEQ ID NO:14).

FIG. 18 also shows the location of the following primers along the toxin B gene: P9 which consists of the sequence 5' CGGAATTCCTAGAAAAAATGGCAA ATG 3' (SEQ ID NO:15); P10 which consists of the sequence 5' GCTCTAGAATGA CCATAAGCTAGCCA 3' (SEQ ID NO:16); P11 which consists of the sequence 5' CGGAATTCGAGTTGGTAGAAAGGTGGA 3' (SEQ ID NO:17); P13 which consists of the sequence 5' CGGAATTCGGTTATTATCTTAAGGATG 3' (SEQ ID NO:18); and P14 which consists of the sequence 5' CGGAATTCTTGATAACTGGAT TTGTGAC 3' (SEQ ID NO:19). The amino acid sequence consisting of amino acid residues 1852 through 2362 of toxin B is listed in SEQ ID NO:20. The amino acid sequence consisting of amino acid residues 1755 through 2362 of toxin B is listed in SEQ ID NO:21. The amino acid sequence consisting of amino acid residues 1754 through 2362 of toxin B is listed in SEQ ID NO:30.

Clostridium difficile VPI strain 10463 was obtained from the American Type Culture Collection (ATCC 43255) and grown under anaerobic conditions in brain-heart infusion medium (Becton Dickinson). High molecular-weight C. difficile DNA was isolated essentially as described Wren and Tabaqchali (1987) J. Clin. Microbiol., 25:2402!, except 1) 100 μg/ml proteinase K in 0.5% SDS was used to disrupt the bacteria and 2) cetytrimethylammonium bromide (CTAB) precipitation as described by Ausubel et al., Eds., Current Protocols in Molecular Biology, Vol. 2 (1989) Current Protocols! was used to remove carbohydrates from the cleared lysate. Briefly, after disruption of the bacteria with proteinase K and SDS, the solution is adjusted to approximately 0.7M NaCl by the addition of a 1/7 volume of 5M NaCl. A 1/10 volume of CTAB/NaCl (10% CTAB in 0.7M NaCl) solution was added and the solution was mixed thoroughly and incubated 10 min at 65° C. An equal volume of chloroform/isoamyl alcohol (24:1) was added and the phases were thoroughly mixed. The organic and aqueous phases were separated by centrifugation in a microfuge for 5 min. The aqueous supernatant was removed and extracted with phenol/chloroform/isoamyl alcohol (25:24:1). The phases were separated by centrifugation in a microfuge for 5 min. The supernatant was transferred to a fresh tube and the DNA was precipitated with isopropanol. The DNA precipitate was pelleted by brief centrifugation in a microfuge. The DNA pellet was washed with 70% ethanol to remove residual CTAB. The DNA pellet was then dried and redissolved in TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). The integrity and yield of genomic DNA was assessed by comparison with a serial dilution of uncut lambda DNA after electrophoresis on an agarose gel.

Toxin B fragments were cloned by PCR utilizing a proofreading thermostable DNA polymerase native Pfu polymerase (Stratagene)!. The high fidelity of this polymerase reduces the mutation problems associated with amplification by error prone polymerases (e.g., Taq polymerase). PCR amplification was performed using the PCR primer pairs P5 (SEQ ID NO:11) with P6 (SEQ ID NO:12) and P7 (SEQ ID NO:13) with P8 (SEQ ID NO:14) in 50 μl reactions containing 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 200 μM of each dNTP, 0.2 μM each primer, and 50 ng C. difficile genomic DNA. Reactions were overlaid with 100 μl mineral oil, heated to 94° C. for 4 min, 0.5 μl native Pfu polymerase (Stratagene) was added, and the reactions were cycled 30 times at 94° C. for 1 min, 50° C. for 1 min, 72° C. (2 min for each kb of sequence to be amplified), followed by 10 min at 72° C. Duplicate reactions were pooled, chloroform extracted, and ethanol precipitated. After washing in 70% ethanol, the pellets were resuspended in 50 μl TE buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA).

The P5/P6 amplification product was cloned into pUC19 as outlined below. 10 μl aliquots of DNA were gel purified using the Prep-a-Gene kit (BioRad), and ligated to SmaI restricted pUC19 vector. Recombinant clones were isolated and confirmed by restriction digestion using standard recombinant molecular biology techniques (Sambrook et al., 1989). Inserts from two independent isolates were identified in which the toxin B insert was oriented such that the vector BamHI and SacI sites were 5' and 3' oriented, respectively (pUCB10-1530). The insert-containing BamHI/SacI fragment was cloned into similarly cut pET23a-c vector DNA, and protein expression was induced in small scale cultures (5 ml) of identified clones.

Total protein extracts were isolated, resolved on SDS-PAGE gels, and toxin B protein identified by Western analysis utilizing a goat anti-toxin B affinity purified antibody (Tech Lab). Procedures for protein induction, SDS-PAGE, and Western blot analysis were performed as described in Williams et al. (1994), supra. In brief, 5 ml cultures of bacteria grown in 2XYT containing 100 μg/ml ampicillin containing the appropriate recombinant clone were induced to express recombinant protein by addition of IPTG to 1 mM. The E. coli hosts used were: BL21(DE3) or BL21(DE3)LysS (Novagen) for pET plasmids.

Cultures were induced by the addition of IPTG to a final concentration of 1.0 mM when the cell density reached 0.5 OD₆₀₀, and induced protein was allowed to accumulate for two hrs after induction. Protein samples were prepared by pelleting 1 ml aliquots of bacteria by centrifugation (1 min in microfuge), and resuspension of the pelleted bacteria in 150 μl of 2X SDS-PAGE sample buffer (0.125 mM Tris-HCl pH 6.8, 2 mM EDTA, 6% SDS, 20% glycerol, 0.025% bromophenol blue; β-mercaptoethanol is added to 5% before use). The samples were heated to 95° C. for 5 min, then cooled and 5 or 10 μls loaded on 7.5% SDS-PAGE gels. High molecular weight protein markers (BioRad) were also loaded, to allow estimation of the MW of identified fusion proteins. After electrophoresis, protein was detected either generally by staining the gels with Coomassie Blue, or specifically, by blotting to nitrocellulose for Western blot detection of specific immunoreactive protein. The MW of induced toxin B reactive protein allowed the integrity of the toxin B reading frame to be determined.

The pET23b recombinant (pPB10-1530) expressed high MW recombinant toxin B reactive protein, consistent with predicted values. This confirmed that frame terminating errors had not occurred during PCR amplification. A pET23b expression clone containing the 10-1750aa interval of the toxin B gene was constructed, by fusion of the EcoRV-SpeI fragment of the P7/P8 amplification product to the P5-EcoRV interval of the P5/P6 amplification product (see FIG. 18) in pPB10-1530. The integrity of this clone (pPB10-1750) was confirmed by restriction mapping, and Western blot detection of expressed recombinant toxin B protein. Levels of induced protein from both pPB10-1530 and pPB10-1750 were too low to facilitate purification of usable amounts of recombinant toxin B protein. The remaining 1750-2360 aa interval was directly cloned into pMAL or pET expression vectors from the P7/P8 amplification product as described below.

b) Expression Of The Toxin B Gene

i) Overview Of Expression Methodologies

Fragments of the toxin B gene were expressed as either native or fusion proteins in E. coli. Native proteins were expressed in either the pET23a-c or pET16b expression vectors (Novagen). The pET23 vectors contain an extensive polylinker sequence in all three reading frames (a-c vectors) followed by a C-terminal poly-histidine repeat. The pET16b vector contains a N-terminal poly-histidine sequence immediately 5' to a small polylinker. The poly-histidine sequence binds to Ni-Chelate columns and allows affinity purification of tagged target proteins Williams et al. (1994), supra!. These affinity tags are small (10 aa for pET16b, 6 aa for pET23) allowing the expression and affinity purification of native proteins with only limited amounts of foreign sequences.

An N-terminal histidine-tagged derivative of pET16b containing an extensive cloning cassette was constructed to facilitate cloning of N-terminal poly-histidine tagged toxin B expressing constructs. This was accomplished by replacement of the promoter region of the pET23a and b vectors with that of the pET16b expression vector. Each vector was restricted with BglII and NdeI, and the reactions resolved on a 1.2% agarose gel. The pET16b promoter region (contained in a 200 bp BglII-NdeI fragment) and the promoter-less pET23 a or b vectors were cut from the gel, mixed and Prep-A-Gene (BioRad) purified. The eluted DNA was ligated, and transformants screened for promoter replacement by NcoI digestion of purified plasmid DNA (the pET16b promoter contains this site, the pET23 promoter does not). These clones (denoted pETHisa or b) contain the pET16b promoter (consisting of a pT7-lac promoter, ribosome binding site and poly-histidine (10 aa) sequence) fused at the NdeI site to the extensive pET23 polylinker.

All MBP fusion proteins were constructed and expressed in the pMAL™-c or pMAL™-p2 vectors (New England Biolabs) in which the protein of interest is expressed as a C-terminal fusion with MBP. All pET plasmids were expressed in either the BL21(DE3) or BL21(DE3)LysS expression hosts, while pMal plasmids were expressed in the BL21 host.

Large scale (500 mls to 1 liter) cultures of each recombinant were grown in 2X YT broth, induced, and soluble protein fractions were isolated as described Williams, et al. (1994), supra!. The soluble protein extracts were affinity chromatographed to isolate recombinant fusion protein, as described Williams et al., (1994) supra!. In brief, extracts containing tagged pET fusions were chromatographed on a nickel chelate column, and eluted using imidazole salts or low pH (pH 4.0) as described by the distributor (Novagen or Qiagen). Extracts containing soluble pMAL fusion protein were prepared and chromatographed in PBS buffer over an amylose resin (New England Biolabs) column, and eluted with PBS containing 10 mM maltose as described Williams et al. (1994), supra!.

ii) Overview Of Toxin B Expression

In both large expression constructs described in (a) above, only low level (i.e., less than 1 mg/liter of intact or nondegraded recombinant protein) expression of recombinant protein was detected. A number of expression constructs containing smaller fragments of the toxin B gene were then constructed, to determine if small regions of the gene can be expressed to high levels (i.e., greater than 1 mg/liter intact protein) without extensive protein degradation. All were constructed by in frame fusions of convenient toxin B restriction fragments to either the pMAL-c, pET23a-c, pET16b or pETHisa-b expression vectors, or by engineering restriction sites at specific locations using PCR amplification using the same conditions described in (a) above!. In all cases, clones were verified by restriction mapping, and, where indicated, DNA sequencing.

Protein preparations from induced cultures of each of these constructs were analyzed, by SDS-PAGE, to estimate protein stability (Coomassie Blue staining) and immunoreactivity against anti-toxin B specific antiserum (Western analysis). Higher levels of intact (i.e., nondegraded), full length fusion proteins were observed with the smaller constructs as compared with the larger recombinants, and a series of expression constructs spanning the entire toxin B gene were constructed (FIGS. 18, 19 and 20 and Table 23).

Constructs that expressed significant levels of recombinant toxin B protein (greater than 1 mg/liter intact recombinant protein) in E. coli were identified and a series of these clones that spans the toxin B gene are shown in FIG. 19 and summarized in Table 23. These clones were utilized to isolate pure toxin B recombinant protein from the entire toxin B gene. Significant protein yields were obtained from pMAL expression constructs spanning the entire toxin B gene, and yields of full length soluble fusion protein ranged from an estimated 1 mg/liter culture (pMB1100-1530) to greater than 20 mg/liter culture (pMB1750-2360).

Representative purifications of MBP and poly-histidine-tagged toxin B recombinants are shown in FIGS. 21 and 22. FIG. 21 shows a Coomassie Blue stained 7.5% SDS-PAGE gel on which various protein samples extracted from bacteria harboring pMB1850-2360 were electrophoresed. Samples were loaded as follows: Lane 1: protein extracted from uninduced culture; Lane 2: induced culture protein; Lane 3: total protein from induced culture after sonication; Lane 4: soluble protein; and Lane 5: eluted affinity purified protein. FIG. 22 depicts the purification of recombinant proteins expressed in bacteria harboring either pPB1850-2360 (Lanes 1-3) or pPB1750-2360 (Lanes 4-6). Samples were loaded as follows: uninduced total protein (Lanes 1 and 4); induced total protein (Lanes 2 and 5); and eluted affinity purified protein (Lanes 3 and 6). The broad range molecular weight protein markers (BioRad) are shown in Lane 7.

Thus, although high level expression was not attained using large expression constructs from the toxin B gene, usable levels of recombinant protein were obtained by isolating induced protein from a series of smaller pMAL expression constructs that span the entire toxin B gene.

These results represent the first demonstration of the feasibility of expressing recombinant toxin B protein to high levels in E. coli. As well, expression of small regions of the putative ligand binding domain (repeat region) of toxin B as native protein yielded insoluble protein, while large constructs, or fusions to MBP were soluble (FIG. 19), demonstrating that specific methodologies are necessary to produce soluble fusion protein from this interval.

iii) Clone Construction And Expression Details

A portion of the toxin B gene containing the toxin B repeat region amino acid residues 1852-2362 of toxin B (SEQ ID NO:20)! was isolated by PCR amplification of this interval of the toxin B gene from C. difficile genomic DNA. The sequence, and location within the toxin B gene, of the two PCR primers P7 (SEQ ID NO: 13) and P8 (SEQ ID NO:14)! used to amplify this region are shown in FIG. 18.

DNA from the PCR amplification was purified by chloroform extraction and ethanol precipitation as described above. The DNA was restricted with SpeI, and the cleaved DNA was resolved by agarose gel electrophoresis. The restriction digestion with SpeI cleaved the 3.6 kb amplification product into a 1.8 kb doublet band. This doublet band was cut from the gel and mixed with appropriately cut, gel purified pMALc or pET23b vector. These vectors were prepared by digestion with HindIII, filling in the overhanging ends using the Klenow enzyme, and cleaving with XbaI (pMALc) or NheI (pET23b). The gel purified DNA fragments were purified using the Prep-A-Gene kit (BioRad) and the DNA was ligated, transformed and putative recombinant clones analyzed by restriction mapping.

pET and pMal clones containing the toxin B repeat insert (aa interval 1750-2360 of toxin B) were verified by restriction mapping, using enzymes that cleaved specific sites within the toxin B region. In both cases fusion of the toxin B SpeI site with either the compatible XbaI site (pMal) or compatible NheI site (pET) is predicted to create an in frame fusion. This was confirmed in the case of the pMB1750-2360 clone by DNA sequencing of the clone junction and 5' end of the toxin B insert using a MBP specific primer (New England Biolabs). In the case of the pET construct, the fusion of the blunt ended toxin B 3' end to the filled HindIII site should create an in-frame fusion with the C-terminal poly-histidine sequence in this vector. The pPB1750-2360 clone selected had lost, as predicted, the HindIII site at this clone junction; this eliminated the possibility that an additional adenosine residue was added to the 3' end of the PCR product by a terminal transferase activity of the Pfu polymerase, since fusion of this adenosine residue to the filled HindIII site would regenerate the restriction site (and was observed in several clones).

One liter cultures of each expression construct were grown, and fusion protein purified by affinity chromatography on either an amylose resin column (pMAL constructs; resin supplied by New England Biolabs) or Ni-chelate column (pET constructs; resin supplied by Qiagen or Novagen) as described Williams et al. (1994), supra!. The integrity and purity of the fusion proteins were determined by Coomassie staining of SDS-PAGE protein gels as well as Western blot analysis with either an affinity purified goat polyclonal antiserum (Tech Lab), or a chicken polyclonal PEG prep, raised against the toxin B protein (CTB) as described above in Example 8. In both cases, affinity purification resulted in yields in excess of 20 mg protein per liter culture, of which greater than 90% was estimated to be full-length recombinant protein. It should be noted that the poly-histidine affinity tagged protein was released from the Qiagen Ni-NTA resin at low imidazole concentration (60 mM), necessitating the use of a 40 mM imidazole rather than a 60 mM imidazole wash step during purification.

A periplasmically secreted version of pMB1750-2360 was constructed by replacement of the promoter and MBP coding region of this construct with that from a related vector (pMAL™-p2; New England Biolabs) in which a signal sequence is present at the N-terminus of the MBP, such that fusion protein is exported. This was accomplished by substituting a BglII-EcoRV promoter fragment from pMAL-p2 into pMB1750-2360. The yields of secreted, affinity purified protein (recovered from osmotic shock extracts as described by Riggs in Current Protocols in Molecular Biology, Vol. 2, Ausubel, et al., Eds. (1989), Current Protocols, pp. 16.6.1-16.6.14! from this vector (pMBp1750-2360) were 6.5 mg/liter culture, of which 50% was estimated to be full-length fusion protein.

The interval was also expressed in two non-overlapping fragments. pMB 1750-1970 was constructed by introduction of a frameshift into pMB 1750-2360, by restriction with HindIII, filling in the overhanging ends and religation of the plasmid. Recombinant clones were selected by loss of the HindIII site, and further restriction map analysis. Recombinant protein expression from this vector was more than 20 mg/liter of greater than 90% pure protein.

The complementary region was expressed in pMB1970-2360. This construct was created by removal of the 1750-1970 interval of pMB1750-2360. This was accomplished by restriction of this plasmid with EcoRI (in the pMalc polylinker 5' to the insert) and HindIII filling in the overhanging ends, and religation of the plasmid. The resultant plasmid, pMB1970-2360, was made using both intracellularly and secreted versions of the pMB1750-2360 vector.

No fusion protein was secreted in the pMB1970-2360 version, perhaps due to a conformational constraint that prevents export of the fusion protein. However, the intracellularly expressed vector produced greater than 40 mg/liter of greater than 90% full-length fusion protein.

Constructs to precisely express the toxin B repeats in either pMalc (pMB1850-2360) or pET16b (pPB1850-2360) were constructed as follows. The DNA interval including the toxin B repeats was PCR amplified as described above utilizing PCR primers P14 (SEQ ID NO:19) and P8 (SEQ ID NO:14). Primer P14 adds a EcoRI site immediately flanking the start of the toxin B repeats.

The amplified fragment was cloned into the pT7 Blue T-vector (Novagen) and recombinant clones in which single copies of the PCR fragment were inserted in either orientation were selected (pT71850-2360) and confirmed by restriction mapping. The insert was excised from two appropriately oriented independently isolated pT71850-2360 plasmids, with EcoRI (5' end of repeats) and PstI (in the flanking polylinker of the vector), and cloned into EcoRI/PstI cleaved pMalc vector. The resulting construct (pMB1850-2360) was confirmed by restriction analysis, and yielded 20 mg/l of soluble fusion protein greater than 90% intact (i.e., nondegraded)! after affinity chromatography. Restriction of this plasmid with HindIII and religation of the vector resulted in the removal of the 1970-2360 interval. The resultant construct (pMB1850-1970) expressed greater than 70 mg/liter of 90% full length fusion protein.

The pPB1850-2360 construct was made by cloning a EcoRI (filled with Klenow)-BamHI fragment from a pT71850-2360 vector (opposite orientation to that used in the pMB1850-2360 construction) into NdeI (filled)/BamHI cleaved pET16b vector. Yields of affinity purified soluble fusion protein were 15 mg/liter, of greater than 90% full length fusion protein.

Several smaller expression constructs from the 1750-2070 interval were also constructed in His-tagged pET vectors, but expression of these plasmids in the BL21 (DE3) host resulted in the production of high levels of mostly insoluble protein (see Table 23 and FIG. 19). These constructs were made as follows.

pPB1850-1970 was constructed by cloning a BglII-HindIII fragment of pPB1850-2360 into BglII/HindIII cleaved pET23b vector. pPB1850-2070 was constructed by cloning a BglII-PvuII fragment of pPB1850-2360 into BglII/HincII cleaved pET23b vector. pPB1750-1970(c) was constructed by removal of the internal HindIII fragment of a pPB1750-2360 vector in which the vector HindIII site was regenerated during cloning (presumably by the addition of an A residue to the amplified PCR product by terminal transferase activity of Pfu polymerase). The pPB1750-1970(n) construct was made by insertion of the insert containing the NdeI-HindIII fragment of pPB1750-2360 into identically cleaved pETHisb vector. All constructs were confirmed by restriction digestion.

An expression construct that directs expression of the 10-470 aa interval of toxin B was constructed in the pMalc vector as follows. A NheI (a site 5' to the insert in the pET23 vector)-AflII (filled) fragment of the toxin B gene from pPB10-1530 was cloned into XbaI (compatible with NheI)/HindIII (filled) pMalc vector. The integrity of the construct (pMB10-470) was verified by restriction mapping and DNA sequencing of the 5' clone junction using a MBP specific DNA primer (New England Biolabs). However, all expressed protein was degraded to the MBP monomer MW.

A second construct spanning this interval (aa 10-470) was constructed by cloning the PCR amplification product from a reaction containing the P9 (SEQ ID NO:15) and P10 (SEQ ID NO:16) primers (FIG. 18) into the pETHisa vector. This was accomplished by cloning the PCR product as an EcoRI-blunt fragment into EcoRI-HincII restricted vector DNA; recombinant clones were verified by restriction mapping. Although this construct (pPB 10-520) allowed expression and purification (utilizing the N-terminal polyhistidine affinity tag) of intact fusion protein, yields were estimated at less than 500 μg per liter culture.

Higher yield of recombinant protein from this interval (aa 10-520) were obtained by expression of the interval in two overlapping clones. The 10-330aa interval was cloned in both pETHisa and pMalc vectors, using the BamHI-AflII (filled) DNA fragment from pPB10-520. This fragment was cloned into BamHI-HindIII (filled) restricted pMalc or BamHI-HincII restricted pETHisa vector. Recombinant clones were verified by restriction mapping. High level expression of either insoluble (pET) or soluble (pMal) fusion protein was obtained. Total yields of fusion protein from the pMB10-330 construct (FIG. 18) were 20 mg/liter culture, of which 10% was estimated to be full-length fusion protein. Although yields of this interval were higher and >90% full-length recombinant protein produced when expressed from the pET construct, the pMal fusion was utilized since the expressed protein was soluble and thus more likely to have the native conformation.

The pMB260-520 clone was constructed by cloning EcoRI-XbaI cleaved amplified DNA from a PCR reaction containing the P11 (SEQ ID NO:17) and P10 (SEQ ID NO:16) DNA primers (FIG. 18) into similarly restricted pMalc vector. Yields of affinity purified protein were 10 mg/liter, of which approximately 50% was estimated to be full-length recombinant protein.

The aa510-1110 interval was expressed as described below. This entire interval was expressed as a pMal fusion by cloning the NheI-HindIII fragment of pUCB10-1530 into XbaI-HindIII cleaved pMalc vector. The integrity of the construct (pMB510-1110) was verified by restriction mapping and DNA sequencing of the 5' clone junction using a MBP specific DNA primer. The yield of affinity purified protein was 25 mg/liter culture, of which 5% was estimated to be full-length fusion protein (1 mg/liter).

To attempt to obtain higher yields, this region was expressed in two fragments (aa510-820, and 820-1110) in the pMalc vector. The pMB510-820 clone was constructed by insertion of a SacI (in the pMalc polylinker 5' to the insert)-HpaI DNA fragment from pMB510-1110 into SacI/StuI restricted pMalc vector. The pMB820-1110 vector was constructed by insertion of the HpaI-HindIII fragment of pUCB10-1530 into BamHI (filled)/HindIII cleaved pMalc vector. The integrity of these constructs were verified by restriction mapping and DNA sequencing of the 5' clone junction using a MBP specific DNA primer.

Recombinant protein expressed from the pMB510-820 vector was highly unstable. However, high levels (20 mg/liter) of >90% full-length fusion protein were obtained from the pMB820-1105 construct. The combination of partially degraded pMB510-1110 protein (enriched for the 510-820 interval) with the pMB820-1110 protein provides usable amounts of recombinant antigen from this interval.

The aa1100-1750 interval was expressed as described below. The entire interval was expressed in the pMalc vector from a construct in which the AccI(filled)-SpeI fragment of pPB10-1750 was inserted into StuI/XbaI (XbaI is compatible with SpeI; StuI and filled AccI sites are both blunt ended) restricted pMalc. The integrity of this construct (pMB1100-1750) was verified by restriction mapping and DNA sequencing of the clone junction using a MBP specific DNA primer. Although 15 mg/liter of affinity purified protein was isolated from cells harboring this construct, the protein was greater than 99% degraded to MBP monomer size.

A smaller derivative of pMB1100-1750 was constructed by restriction of pMB1100-1750 with AflII and SalI (in the pMalc polylinker 3' to the insert), filling in the overhanging ends, and religating the plasmid. The resultant clone (verified by restriction digestion and DNA sequencing) has deleted the aa1530-1750 interval, was designated pMB1100-1530. pMB1100-1530 expressed recombinant protein at a yield of greater than 40 mg/liter, of which 5% was estimated to be full-length fusion protein.

Three constructs were made to express the remaining interval. Initially, a BspHI (filled)-SpeI fragment from pPB10-1750 was cloned into EcoRI(filled)/XbaI cleaved pMalc vector. The integrity of this construct (pMB1570-1750) was verified by restriction mapping and DNA sequencing of the 5' clone junction using a MBP specific DNA primer. Expression of recombinant protein from this plasmid was very low, approximately 3 mg affinity purified protein per liter, and most was degraded to MBP monomer size. This region was subsequently expressed from a PCR amplified DNA fragment. A PCR reaction utilizing primers P13 SEQ ID NO: 18; P13 was engineered to introduce an EcoRI site 5' to amplified toxin B sequences! and P8 (SEQ ID NO:14) was performed on C. difficile genomic DNA as described above. The amplified fragment was cleaved with EcoRI and SpeI, and cloned into EcoRI/XbaI cleaved pMalc vector. The resultant clone (pMB1530-1750) was verified by restriction map analysis, and recombinant protein was expressed and purified. The yield was greater than 20 mg protein per liter culture and it was estimated that 25% was full-length fusion protein; this was a significantly higher yield than the original pMB1570-1750 clone. The insert of pMB1530-1750 (in a EcoR-SalI fragment) was transferred to the pETHisa vector (EcoRI/XhoI cleaved, XhoI and SalI ends are compatible). No detectable fusion protein was purified on Ni-Chelate columns from soluble lysates of cells induced to express fusion protein from this construct.

                  TABLE 23                                                         ______________________________________                                         Summary Of Toxin B Expression Constructs.sup.a                                 Clone     Affinity Tag                                                                             Yield (mg/liter)                                                                             % Full Length                                ______________________________________                                         pPB10-1750                                                                               none      low (estimated from                                                                          ?                                                                Western blot hyb.)                                         pPB10-1530                                                                               none      low (as above)                                                                               ?                                            pMB10-470 MBP       15 mg/l        0%                                          pPB10-520 poly-his  0.5 mg/l      20%                                          pPB10-330 poly-his  >20 mg/l (insoluble)                                                                         90%                                          pMB10-330 MBP       20 mg/l       10%                                          pMB260-520                                                                               MBP       10 mg/l       50%                                          pMB510-1110                                                                              MBP       25 mg/l       5%                                           pMB510-820                                                                               MBP       degraded (by                                                                   Western blot hyb)                                          pMB820-1110                                                                              MBP       20 mg/l       90%                                          pMB1100-1750                                                                             MBP       15 mg/l       0%                                           pMB1100-1530                                                                             MBP       40 mg/l       5%                                           pMB1570-1750                                                                             MBP       3 mg/l        <5%                                          pPB1530-1750                                                                             poly-his  no purified   ?                                                                protein detected                                           pMB1530-1750                                                                             MBP       20 mg/l       25%                                          pMB1750-2360                                                                             MBP       >20 mg/l      >90%                                         pMB1750-2360                                                                             MBP       6.5 mg/l (secreted)                                                                          50%                                          pPB1750-2360                                                                             poly-his  >20 mg/l      >90%                                         pMB1750-1970                                                                             MBP       >20 mg/l      >90%                                         pMB1970-2360                                                                             MBP       40 mg/l       >90%                                         pMB1970-2360                                                                             MBP       (no secretion)                                                                               NA                                           pMB1850-2360                                                                             MBP       20 mg/l       >90%                                         pPB1850-2360                                                                             poly-his  15 mg/l       >90%                                         pMB1850-1970                                                                             MBP       70 mg/l       >90%                                         pPB1850-1970                                                                             poly-his  >10 mg/l (insoluble)                                                                         >90%                                         pPB1850-2070                                                                             poly-his  >10 mg/l (insoluble)                                                                         >90%                                         pPB1750-1970(c)                                                                          poly-his  >10 mg/l (insoluble)                                                                         >90%                                         pPB1750-1970(n)                                                                          poly-his  >10 mg/l (insoluble)                                                                         >90%                                         ______________________________________                                          .sup.a Clones in italics are clones currently utilized to purify               recombinant protein from each selected interval.                         

EXAMPLE 19 Identification, Purification And Induction Of Neutralizing Antibodies Against Recombinant C. difficile Toxin B Protein

To determine whether recombinant toxin B polypeptide fragments can generate neutralizing antibodies, typically animals would first be immunized with recombinant proteins and anti-recombinant antibodies are generated. These anti-recombinant protein antibodies are then tested for neutralizing ability in vivo or in vitro. Depending on the immunogenic nature of the recombinant polypeptide, the generation of high-titer antibodies against that protein may take several months. To accelerate this process and identify which recombinant polypeptide(s) may be the best candidate to generate neutralizing antibodies, depletion studies were performed. Specifically, recombinant toxin B polypeptide were pre-screened by testing whether they have the ability to bind to protective antibodies from a CTB antibody preparation and hence deplete those antibodies of their neutralizing capacity. Those recombinant polypeptides found to bind CTB, were then utilized to generate neutralizing antibodies. This Example involved: a) identification of recombinant sub-regions within toxin B to which neutralizing antibodies bind; b) identification of toxin B sub-region specific antibodies that neutralize toxin B in vivo; and c) generation and evaluation of antibodies reactive to recombinant toxin B polypeptides.

a) Identification Of Recombinant Sub-Regions Within Toxin B To Which Neutralizing Antibodies Bind

Sub-regions within toxin B to which neutralizing antibodies bind were identified by utilizing recombinant toxin B proteins to deplete protective antibodies from a polyclonal pool of antibodies against native C. difficile toxin B. An in vivo assay was developed to evaluate protein preparations for the ability to bind neutralizing antibodies. Recombinant proteins were first pre-mixed with antibodies directed against native toxin B (CTB antibody; see Example 8) and allowed to react for one hour at 37° C. Subsequently, C. difficile toxin B (CTB; Tech Lab) was added at a concentration lethal to hamsters and incubated for another hour at 37° C. After incubation this mixture was injected intraperitoneally (IP) into hamsters. If the recombinant polypeptide contains neutralizing epitopes, the CTB antibodies will lose its ability to protect the hamsters against death from CTB. If partial or complete protection occurs with the CTB antibody-recombinant mixture, that recombinant contains only weak or non-neutralizing epitopes of toxin B. This assay was performed as follows.

Antibodies against CTB were generated in egg laying Leghorn hens as described in Example 8. The lethal dosage (LD₁₀₀) of C. difficile toxin B when delivered I.P. into 40 g female Golden Syrian hamsters (Charles River) was determined to be 2.5 to 5 μg. Antibodies generated against CTB and purified by PEG precipitation could completely protect the hamsters at the I.P. dosage determined above. The minimal amount of CTB antibody needed to afford good protection against 5 μg of CTB when injected I.P. into hamsters was also determined (1X PEG prep). These experiments defined the parameters needed to test whether a given recombinant protein could deplete protective CTB antibodies.

The cloned regions tested for neutralizing ability cover the entire toxin B gene and were designated as Intervals (INT) 1 through 5 (see FIG. 19). Approximately equivalent final concentrations of each recombinant polypeptide were tested. The following recombinant polypeptides were used: 1) a mixture of intervals 1 and 2 (INT-1, 2); 2) a mixture of Intervals 4 and 5 (INT-4, 5) and 3) Interval 3 (INT-3). Recombinant proteins (each at about 1 mg total protein) were first preincubated with a final CTB antibody concentration of 1X i.e., pellet dissolved in original yolk volume as described in Example 1 (c)! in a final volume of 5 mls for 1 hour at 37° C. Twenty-five μg of CTB (at a concentration of 5 μg/ml), enough CTB to kill 5 hamsters, was then added and the mixture was then incubated for 1 hour at 37° C. Five, 40 g female hamsters (Charles River) in each treatment group were then each given 1 ml of the mixture I.P. using a tuberculin syringe with a 27 gauge needle. The results of this experiment are shown in Table 24.

                  TABLE 24                                                         ______________________________________                                         Binding Of Neutralizing Antibodies By INT 3 Protein                                          Number of    Number of                                           Treatment Group.sup.1                                                                        Animals Alive                                                                               Animals Dead                                        ______________________________________                                         CTB antibodies                                                                               3            2                                                   CTB antibodies + INT1,2                                                                      3            2                                                   CTB antibodies + INT4,5                                                                      3            2                                                   CTB antibodies + INT 3                                                                       0            5                                                   ______________________________________                                          .sup.1 C. difficile toxin B (CTB) was added to each group.               

As shown in Table 24, the addition of recombinant proteins from INT-1, 2 or INT-4, 5 had no effect on the in vivo protective ability of the CTB antibody preparation compared to the CTB antibody preparation alone. In contrast, INT-3 recombinant polypeptide was able to remove all of the toxin B neutralizing ability of the CTB antibodies as demonstrated by the death of all the hamsters in that group.

The above experiment was repeated, using two smaller expressed fragments (pMB 1750-1970 and pMB 1970-2360, see FIG. 19) comprising the INT-3 domain to determine if that domain could be further subdivided into smaller neutralizing epitopes. In addition, full-length INT-3 polypeptide expressed as a histidine tagged protein (pPB1750-2360) was tested for neutralizing ability and compared to the original INT-3 expressed MBP fusion (pMB1750-2360). The results are shown in Table 25.

                  TABLE 25                                                         ______________________________________                                         Removal Of Neutralizing Antibodies By Repeat Containing Proteins                                Number Of   Number Of                                         Treatment Group.sup.1                                                                           Animals Alive                                                                              Animals Dead                                      ______________________________________                                         CTB antibodies   5           0                                                 CTB antibodies + pPB1750-2360                                                                   0           5                                                 CTB antibodies + pMB1750-2360                                                                   0           5                                                 CTB antibodies + pMB1970-2360                                                                   3           2                                                 CTB antibodies + pMB1750-1970                                                                   2           3                                                 ______________________________________                                          .sup.1 C. difficile toxin B (CTB) was added to each group.               

The results summarized in Table 25 indicate that the smaller polypeptide fragments within the INT-3 domain, pMB1750-1970 and pMB1970-2360, partially lose the ability to bind to and remove neutralizing antibodies from the CTB antibody pool. These results demonstrate that the full length INT-3 polypeptide is required to completely deplete the CTB antibody pool of neutralizing antibodies. This experiment also shows that the neutralization epitope of INT-3 can be expressed in alternative vector systems and the results are independent of the vector utilized or the accompanying fusion partner.

Other Interval 3 specific proteins were subsequently tested for the ability to remove neutralizing antibodies within the CTB antibody pool as described above. The Interval 3 specific proteins used in these studies are summarized in FIG. 23. In FIG. 23 the following abbreviations are used: pP refers to the pET23 vector; pM refers to the pMALc vector; B refers to toxin B; the numbers refer to the amino acid interval expressed in the clone. The solid black ovals represent the MBP; and HHH represents the poly-histidine tag.

Only recombinant proteins comprising the entire toxin B repeat domain (pMB1750-2360, pPB1750-2360 and pPB1850-2360) can bind and completely remove normalizing antibodies from the CTB antibody pool. Recombinant proteins comprising only a portion of the toxin B repeat domain were not capable of completely removing neutralizing antibodies from the CTB antibody pool (pMB1750-1970 and pMB1970-2360 could partially remove neutralizing antibodies while pMB1850-1970 and pPB1850-2070 failed to remove any neutralizing antibodies from the CTB antibody pool).

The above results demonstrate that only the complete ligand binding domain (repeat region) of the toxin B gene can bind and completely remove neutralizing antibodies from the CTB antibody pool. These results demonstrate that antibodies directed against the entire toxin B repeat region are necessary for in vivo toxin neutralization (see FIG. 23; only the recombinant proteins expressed by the pMB1750-2360, pPB1750-2360 and pPB1850-2360 vectors are capable of completely removing the neutralizing antibodies from the CTB antibody pool).

These results represent the first indication that the entire repeat region of toxin B would be necessary for the generation of antibodies capable of neutralizing toxin B, and that sub-regions may not be sufficient to generate maximal titers of neutralizing antibodies.

b) Identification Of Toxin B Sub-Region Specific Antibodies That Neutralize Toxin B In Vivo

To determine if antibodies directed against the toxin B repeat region are sufficient for neutralization, region specific antibodies within the CTB antibody preparation were affinity purified, and tested for in vivo neutralization. Affinity colurines containing recombinant toxin B repeat proteins were made as described below. A separate affinity column was prepared using each of the following recombinant toxin B repeat proteins: pPB1750-2360, pPB1850-2360, pMB1750-1970 and pMB 1970-2360.

For each affinity column to be made, four ml of PBS-washed Actigel resin (Sterogene) was coupled overnight at room temperature with 5-10 mg of affinity purified recombinant protein (first extensively dialyzed into PBS) in 15 ml tubes (Falcon) containing a 1/10 final volume Ald-coupling solution (1M sodium cyanoborohydride). Aliquots of the supernatants from the coupling reactions, before and after coupling, were assessed by Coomassie staining of 7.5% SDS-PAGE gels. Based on protein band intensities, in all cases greater than 30% coupling efficiencies were estimated. The resins were poured into 10 ml columns (BioRad), washed extensively with PBS, pre-eluted with 4M guanidine-HCl (in 10 mM Tris-HCl, pH 8.0) and reequilibrated in PBS. The columns were stored at 4° C.

Aliquots of a CTB IgY polyclonal antibody preparation (PEG prep) were affinity purified on each of the four columns as described below. The columns were hooked to a UV monitor (ISCO), washed with PBS and 40 ml aliquots of a 2X PEG prep (filter sterilized using a 0.45μ filter) were applied. The columns were washed with PBS until the baseline value was re-established. The columns were then washed with BBStween to elute nonspecifically binding antibodies, and reequilibrated with PBS. Bound antibody was eluted from the column in 4M guanidine-HCl (in 10 mM Tris-HCl, pH 8.0). The eluted antibody was immediately dialyzed against a 100-fold excess of PBS at 4° C. for 2 hrs. The samples were then dialyzed extensively against at least 2 changes of PBS, and affinity purified antibody was collected and stored at 4° C. The antibody preparations were quantified by UV absorbance. The elution volumes were in the range of 4-8 ml. All affinity purified stocks contained similar total antibody concentrations, ranging from 0.25-0.35% of the total protein applied to the columns.

The ability of the affinity purified antibody preparations to neutralize toxin B in vivo was determined using the assay outlined in a) above. Affinity purified antibody was diluted 1:1 in PBS before testing. The results are shown in Table 26.

In all cases similar levels of toxin neutralization was observed, such that lethality was delayed in all groups relative to preimmune controls. This result demonstrates that antibodies reactive to the repeat region of the toxin B gene are sufficient to neutralize toxin B in vivo. The hamsters will eventually die in all groups, but this death is maximally delayed with the CTB PEG prep antibodies. Thus neutralization with the affinity purified (AP) antibodies is not as complete as that observed with the CTB prep before affinity chromatography. This result may be due to loss of activity during guanidine denaturation (during the elution of the antibodies from the affinity column) or the presence of antibodies specific to other regions of the toxin B gene that can contribute to toxin neutralization (present in the CTB PEG prep).

                  TABLE 26                                                         ______________________________________                                         Neutralization Of Toxin B By Affinity Purified Antibodies                                       Number Of   Number Of                                         Treatment Group.sup.a                                                                           Animals Alive.sup.b                                                                        Animals Dead.sup.b                                ______________________________________                                         Preimmune.sup.1  0           5                                                 CTB.sup.1 ; 400 μg                                                                           5           0                                                 CTB (AP on pPB1750-2360);.sup.2 875 μg                                                       5           0                                                 CTB (AP on pMB1750-1970);.sup.2 875 μg                                                       5           0                                                 CTB (AP on pMB1970-2360);.sup.2 500 μg                                                       5           0                                                 ______________________________________                                          .sup.a C. difficile toxin B (CTB) (Tech Lab; at 5 μg/ml, 25 μg           total) at lethal concentration to hamsters is added to antibody and            incubated for one hour at 37° C. After incubation this mixture is       injected intraperitoneally (IP) into hamsters. Each treatment group            received toxin premixed with antibody raised against the indicated             protein, as either: .sup.1 4X antibody PEG prep or .sup.2 affinity             purified (AP) antibody (from CTB PEG prep, on indicated columns). The          amount of specific antibody in each prep is indicated; the amount is           directly determined for affinity purified preps and is estimated for the       4X CTB as described in Example 15.                                             .sup.b The numbers in each group represent numbers of hamsters dead or         alive, 2 hr post IP administration of toxin/antibody mixture.            

The observation that antibodies affinity purified against the non-overlapping pMB1750-1970 and pMB1970-2360 proteins neutralized toxin B raised the possibility that either 1) antibodies specific to repeat sub-regions are sufficient to neutralize toxin B or 2) sub-region specific proteins can bind most or all repeat specific antibodies present in the CTB polyclonal pool. This would likely be due to conformational similarities between repeats, since homology in the primary amino acid sequences between different repeats is in the range of only 25-75% Eichel-Streiber, et al. (1992) Molec. Gen. Genetics 233:260!. These possibilities were tested by affinity chromatography.

The CTB PEG prep was sequentially depleted 2X on the pMB1750-1970 column; only a small elution peak was observed after the second chromatography, indicating that most reactive antibodies were removed. This interval depleted CTB preparation was then chromatographed on the pPB1850-2360 column; no antibody bound to the column. The reactivity of the CTB and CTB (pMB1750-1970 depleted) preps to pPB1750-2360, pPB1850-2360, pMB1750-1970 and pMB1970-2360 proteins was then determined by ELISA using the protocol described in Example 13(c). Briefly, 96-well microtiter plates (Falcon, Pro-Bind Assay Plates) were coated with recombinant protein by adding 100 μl volumes of protein at 1-2 μg/ml in PBS containing 0.005% thimerosal to each well and incubating overnight at 4° C. The next morning, the coating suspensions were decanted and the wells were washed three times using PBS. In order to block non-specific binding sites, 100 μl of 1.0% BSA (Sigma) in PBS (blocking solution) was then added to each well, and the plates were incubated for 1 hr. at 37° C. The blocking solution was decanted and duplicate samples of 150 μl of diluted antibody was added to the first well of a dilution series. The initial testing serum dilution was (1/200 for CTB prep, (the concentration of depleted CTB was standardized by OD₂₈₀) in blocking solution containing 0.5% Tween 20, followed by 5-fold serial dilutions into this solution. This was accomplished by serially transferring 30 μl aliquots to 120 μl buffer, mixing, and repeating the dilution into a fresh well. After the final dilution, 30 μl was removed from the well such that all wells contained 120 μl final volume. A total of 5 such dilutions were performed (4 wells total). The plates were incubated for 1 hr at 37° C. Following this incubation, the serially diluted samples were decanted and the wells were washed three times using PBS containing 0.5% Tween 20 (PBST), followed by two 5 min washes using BBS-Tween and a final three washes using PBST. To each well, 100 μl of 1/1000 diluted secondary antibody rabbit anti-chicken IgG alkaline phosphatase (Sigma) diluted in blocking solution containing 0.5% Tween 20! was added, and the plate was incubated 1 hr at 37° C. The conjugate solutions were decanted and the plates were washed 6 times in PBST, then once in 50 mM Na₂ CO₃, 10 mM MgCl₂, pH 9.5. The plates were developed by the addition of 100 μl of a solution containing 1 mg/ml para-nitro phenyl phosphate (Sigma) dissolved in 50 mM Na₂ CO₃, 10 mM MgCl₂, pH 9.5 to each well. The plates were then incubated at room temperature in the dark for 5-45 min. The absorbency of each well was measured at 410 nm using a Dynatech MR 700 plate reader.

As predicted by the affinity chromatography results, depletion of the CTB prep on the pMB1750-1970 column removed all detectable reactivity to the pMB1970-2360 protein. The reciprocal purification of a CTB prep that was depleted on the pMB1970-2360 column yielded no bound antibody when chromatographed on the pMB1750-1970 column. These results demonstrate that all repeat reactive antibodies in the CTB polyclonal pool recognize a conserved structure that is present in non-overlapping repeats. Although it is possible that this conserved structure represents rare conserved linear epitopes, it appears more likely that the neutralizing antibodies recognize a specific protein conformation. This conclusion was also suggested by the results of Western blot hybridization analysis of CTB reactivity to these recombinant proteins.

Western blots of 7.5% SDS-PAGE gels, loaded and electrophoresed with defined quantities of each recombinant protein, were probed with the CTB polyclonal antibody preparation. The blots were prepared and developed with alkaline phosphatase as described in Example 3. The results are shown in FIG. 24.

FIG. 24 depicts a comparison of immunoreactivity of IgY antibody raised against either native or recombinant toxin B antigen. Equal amounts of pMB1750-1970 (lane 1), pMB1970-2360 (lane 2), pPB1850-2360 (lane 3) as well as a serial dilution of pPB1750-2360 (lanes 4-6 comprising 1X, 1/10X and 1/100X amounts, respectively) proteins were loaded in duplicate and resolved on a 7.5% SDS-PAGE gel. The gel was blotted and each half was hybridized with PEG prep IgY antibodies from chickens immunized with either native CTB or pPB1750-2360 protein. Note that the full-length pMB1750-1970 protein was identified only by antibodies reactive to the recombinant protein (arrows).

Although the CTB prep reacts with the pPB1750-2360, pPB1850-2360, and pMB 1970-2360 proteins, no reactivity to the pMB 1750-1970 protein was observed (FIG. 24). Given that all repeat reactive antibodies can be bound by this protein during affinity chromatography, this result indicates that the protein cannot fold properly on Western blots. Since this eliminates all antibody reactivity, it is unlikely that the repeat reactive antibodies in the CTB prep recognize linear epitopes. This may indicate that in order to induce protective antibodies, recombinant toxin B protein will need to be properly folded.

e) Generation And Evaluation Of Antibodies Reactive To Recombinant Toxin B Polypeptides

i) Generation Of Antibodies Reactive To Recombinant Toxin B Proteins

Antibodies against recombinant proteins were generated in egg laying Leghorn hens as described in Example 13. Antibodies were raised using Freund's adjuvant (Gibco) unless otherwise indicated! against the following recombinant proteins: 1) a mixture of Interval 1+2 proteins (see FIG. 18); 2) a mixture of interval 4 and 5 proteins (see FIG. 18); 3)pMB1970-2360 protein; 4)pPB1750-2360 protein; 5) pMB1750-2360; 6) pMB1750-2360 Titermax adjuvant (Vaxcell)!; 7) pMB1750-2360 Gerbu adjuvant (Biotech)!; 8) pMBp1750-2360 protein; 9) pPB1850-2360; and 10) pMB1850-2360.

Chickens were boosted at least 3 times with recombinant protein until ELISA reactivity using the protocol described in b) above with the exception that the plates were coated with pPB1750-2360 protein! of polyclonal PEG preps was at least equal to that of the CTB polyclonal antibody PEG prep. ELISA titers were determined for the PEG preps from all of the above immunogens and were found to be comparable ranging from 1:12500 to 1:62500. High titers were achieved in all cases except in 6) pMB1750-2360 in which strong titers were not observed using the Titermax adjuvant, and this preparation was not tested further.

ii) Evaluation Of Antibodies Reactive To Recombinant Proteins By Western Blot Hybridization

Western blots of 7.5% SDS-PAGE gels, loaded and electrophoresed with defined quantities of recombinant protein (pMB 1750-1970, pPB 1850-2360, and pMB1970-2360 proteins and a serial dilution of the pPB1750-2360 to allow quantification of reactivity), were probed with the CTB, pPB1750-2360, pMB1750-2360 and pMB 1970-2360 polyclonal antibody preparations (from chickens immunized using Freund's adjuvant). The blots were prepared and developed with alkaline phosphatase as described above in b).

As shown in FIG. 24, the CTB and pMB1970-2360 preps reacted strongly with the pPB1750-2360, pPB1850-2360, and pMB1970-2360 proteins while the pPB1750-2360 and pMB1970-2360 (Gerbu) preparations reacted strongly with all four proteins. The Western blot reactivity of the pPB1750-2360 and pMB1970-2360 (Gerbu) preparations were equivalent to that of the CTB preparation, while reactivity of the pMB1970-2360 preparation was <10% that of the CTB prep. Despite equivalent ELISA reactivities only weak reactivity (approximately 1%) to the recombinant proteins were observed in PEG preps from two independent groups immunized with the pMB1750-2360 protein and one group immunized with the pMB1750-2360 preparation using Freund's adjuvant.

Affinity purification was utilized to determine if this difference in immunoreactivity by Western blot analysis reflects differing antibody titers. Fifty ml 2X PEG preparations from chickens immunized with either pMB1750-2360 or pMB1970-2360 protein were chromatographed on the pPB 1750-2360 affinity column from b) above, as described. The yield of affinity purified antibody (% total protein in preparation) was equivalent to the yield obtained from a CTB PEG preparation in b) above. Thus, differences in Western reactivity reflect a qualitative difference in the antibody pools, rather than quantitative differences., These results demonstrate that certain recombinant proteins are more effective at generating high affinity antibodies (as assayed by Western blot hybridization).

iii) In Vivo Neutralization Of Toxin B Using Antibodies Reactive To Recombinant Protein

The in vivo hamster model described in Examples 9 and 14(b)! was utilized to assess the neutralizing ability of antibodies raised against recombinant toxin B proteins. The results from three experiments are shown below in Tables 27-29.

The ability of each immunogen to neutralize toxin B in vivo has been compiled and is shown in Table 30. As predicted from the recombinant protein-CTB premix studies (Table 24) only antibodies to Interval 3 (1750-2366) and not the other regions of toxin B (i.e., intervals 1-5) are protective. Unexpectedly, antibodies generated to INT-3 region expressed in pMAL vector (pMB1750-2360 and pMB1750-2360) using Freund's adjuvant were non-neutralizing. This observation is reproducible, since no neutralization was observed in two independent immunizations with pMB 1750-2360 and one immunization with pMpB 1750-2360. The fact that 5X quantities of affinity purified toxin B repeat specific antibodies from pMB1750-2360 PEG preps cannot neutralize toxin B while 1X quantities of affinity purified anti-CTB antibodies can (Table 28) demonstrates that the differential ability of CTB antibodies to neutralize toxin B is due to qualitative rather than quantitative differences in these antibody preparations. Only when this region was expressed in an alternative vector (pPB1750-2360) or using an alternative adjuvant with the pMB1750-2360 protein were neutralizing antibodies generated. Importantly, antibodies raised using Freund's adjuvant to pPB 1850-2360, which contains a fragment that is only 100 amino acids smaller than recombinant pPB1750-2360, are unable to neutralize toxin B in vivo (Table 27); note also that the same vector is used for both pPB1850-2360 and pPB1750-2360.

                  TABLE 27                                                         ______________________________________                                         In Vivo Neutralization Of Toxin B                                              Treatment Group.sup.a                                                                     Number Animals Alive.sup.b                                                                    Number Animals Dead.sup.b                            ______________________________________                                         Preimmune  0              5                                                    CTB        5              0                                                    INT 1+2    0              5                                                    INT 4+5    0              5                                                    pMB1750-2360                                                                              0              5                                                    pMB1970-2360                                                                              0              5                                                    pPB1750-2360                                                                              5              0                                                    ______________________________________                                          .sup.a C. difficile toxin B (CTB) (at 5 μg/ml; 25 μg total; Tech         Lab) at lethal concentration to hamsters is added to antibody and              incubated for one hour at 37° C. After incubation this mixture is       injected intraperitoneally (IP) into hamsters. Each treatment group            received toxin premixed with antibody raised against the indicated             protein, as a 4X antibody PEG prep.                                            .sup.b The numbers in each group represent numbers of hamsters dead or         alive, 2 hours post IP administration of toxin/antibody mixture.         

                  TABLE 28                                                         ______________________________________                                         In Vivo Neutralization Of Toxin B Using Affinity Purified Antibodies                            Number Animals                                                                             Number Animals                                    Treatment Group.sup.a                                                                           Alive.sup.b Dead.sup.b                                        ______________________________________                                         Preimmune(1)     0           5                                                 CTB(1)           5           0                                                 pPB1750-2360(1)  5           0                                                 1.5 mg anti-pMB1750-2360(2)                                                                     1           4                                                 1.5 mg anti-pMB1970-2360(2)                                                                     0           5                                                 300 μg anti-CTB(2)                                                                           5           0                                                 ______________________________________                                          .sup.a C. difficile toxin B (CTB) (at 5 μg/ml; 25 μg total; Tech         Lab) at lethal concentration to hamsters is added to antibody and              incubated for one hour at 37° C. After incubation, 1 ml of this         mixture is injected intraperitoneally (IP) into hamsters. Each treatment       group received toxin premixed with antibody raised against the indicated       protein, as either (1) 4X antibody PEG prep or (2) affinity purified           antibody (on a pBP17502360 resin), either 1.5 mg/group (antipMB1750-2360       and antipMB1970-2360; used undiluted affinity purified antibody) or 350        μg/group (antiCTB, repeat specific; used 1/5 diluted antiCTB antibody)      .sup.b The numbers in each group represent numbers of hamsters dead or         alive, 2 hr postIP administration of toxin/antibody mixture.             

                  TABLE 29                                                         ______________________________________                                         Generation Of Neutralizing Antibodies Utilizing The Gerbu Adjuvant             Treatment Group.sup.a                                                                     Number Animals Alive.sup.b                                                                    Number Animals Dead.sup.b                            ______________________________________                                         Preimmune  0              5                                                    CTB        5              0                                                    pMB1970-2360                                                                              0              5                                                    pMB1850-2360                                                                              0              5                                                    pPB1850-2360                                                                              0              5                                                    pMB1750-2360                                                                              5              0                                                    (Gerbu adj)                                                                    ______________________________________                                          .sup.a C. difficile toxin B (CTB) (Tech Lab) at lethal concentration to        hamsters is added to antibody and incubated for one hour at 37° C.      After incubation this mixture is injected intraperitoneally (IP) into          hamsters. Each treatment group received toxin premixed with antibody           raised against the indicated protein, as a 4X antibody PEG prep.               .sup.b The numbers in each group represent numbers of hamsters dead or         alive, 2 hrs post IP administration of toxin/antibody mixture.           

                                      TABLE 30                                     __________________________________________________________________________     In Vivo Neutralization Of Toxin B                                                               Tested Antigen In vivo                                        Immunogen Adjuvant                                                                              Preparation.sup.a                                                                     Utilized For AP                                                                        Neutralization.sup.b                           __________________________________________________________________________     Preimmune NA.sup.1                                                                              PEG    NA      no                                             CTB (native)                                                                             Titermax                                                                              PEG    NA      yes                                            CTB (native)                                                                             Titermax                                                                              AP     pPB1750-2360                                                                           yes                                            CTB (native)                                                                             Titermax                                                                              AP     pPB1850-2360                                                                           yes                                            CTB (native)                                                                             Titermax                                                                              AP     pPB1750-1970                                                                           yes                                            CTB (native)                                                                             Titermax                                                                              AP     pPB1970-2360                                                                           yes                                            pMB1750-2360                                                                             Freunds                                                                               PEG    NA      no                                             pMB1750-2360                                                                             Freunds                                                                               AP     pPB1750-2360                                                                           no                                             pMB1750-2360                                                                             Gerbu  PEG    NA      yes                                            pMB1970-2360                                                                             Freunds                                                                               PEG    NA      no                                             pMB1970-2360                                                                             Freunds                                                                               AP     pPB1750-2360                                                                           no                                             pPB1750-2360                                                                             Freunds                                                                               PEG    NA      yes                                            pPB1850-2360                                                                             Freunds                                                                               PEG    NA      no                                             pMB1850-2360                                                                             Freunds                                                                               PEG    NA      no                                             INT 1 + 2 Freunds                                                                               PEG    NA      no                                             INT 4 + 5 Freunds                                                                               PEG    NA      no                                             __________________________________________________________________________      .sup.a Either PEG preparation (PEG) or affinity purified antibodies (AP).      .sup.b `Yes` denotes complete neutralization (0/5 dead) while `no` denote      no neutralization (5/5 dead) of toxin B, 2 hours postadministration of         mixture.                                                                       .sup.1 `NA` denotes not applicable.                                      

The pPB1750-2360 antibody pool confers significant in vivo protection, equivalent to that obtained with the affinity purified CTB antibodies. This correlates with the observed high affinity of this antibody pool (relative to the pMB1750-2360 or pMB1970-2360 pools) as assayed by Western blot analysis (FIG. 24). These results provide the first demonstration that in vivo neutralizing antibodies can be induced using recombinant toxin B protein as immunogen.

The failure of high concentrations of antibodies raised against the pMB1750-2360 protein (using Freunds adjuvant) to neutralize, while the use of Gerbu adjuvant and pMB 1750-2360 protein generates a neutralizing response, demonstrates that conformation or presentation of this protein is essential for the induction of neutralizing antibodies. These results are consistent with the observation that the neutralizing antibodies produced when native CTB is used as an immunogen appear to recognize conformational epitopes see section b) above!. This is the first demonstration that the conformation or presentation of recombinant toxin B protein is essential to generate high titers of neutralizing antibodies.

EXAMPLE 20 Determination Of Quantitative And Qualitative Differences Between pMB 1750-2360, pMB 1750-2360 (Gerbu) Or pPB1750-2360 IgY Polyclonal Antibody Preparations

In Example 19, it was demonstrated that toxin B neutralizing antibodies could be generated using specific recombinant toxin B proteins (pPB1750-2360) or specific adjuvants. Antibodies raised against pMB1750-2360 were capable of neutralizing the enterotoxic effect of toxin B when the recombinant protein was used to immunize hens in conjunction with the Gerbu adjuvant, but not when Freunds adjuvant was used. To determine the basis for these antigen and adjuvant restrictions, toxin B-specific antibodies present in the neutralizing and non-neutralizing PEG preparations were isolated by affinity chromatography and tested for qualitative or quantitative differences. The example involved a) purification of anti-toxin B specific antibodies from pMB1750-2360 and pPB1750-2360 PEG preparations and b) in vivo neutralization of toxin B using the affinity purified antibody.

a) Purification Of Specific Antibodies From pMB1750-2360 And pPB1750-2360 PEG Preparations

To purify and determine the concentration of specific antibodies (expressed as the percent of total antibody) within the pPB1750-2360 (Freunds and Gerbu) and pPB1750-2360 PEG preparations, defined quantities of these antibody preparations were chromatographed on an affinity column containing the entire toxin B repeat region (pPB1750-2360). The amount of affinity purified antibody was then quantified.

An affinity column containing the recombinant toxin B repeat protein, pPB1750-2360, was made as follows. Four ml of PBS-washed Actigel resin (Sterogene) was coupled with 5 mg of pPB1750-2360 affinity purified protein (dialyzed into PBS; estimated to be greater than 95% full length fusion protein) in a 15 ml tube (Falcon) containing 1/10 final volume Ald-coupling solution (1M sodium cyanoborohydride). Aliquots of the supernatant from the coupling reactions, before and after coupling, were assessed by Coomassie staining of 7.5% SDS-PAGE gels. Based on protein band intensities, greater than 95% (approximately 5 mg) of recombinant protein was coupled to the resin. The coupled resin was poured into a 10 ml column (BioRad), washed extensively with PBS, pre-eluted with 4M guanidine-HCl (in 10 mM Tris-HCL, pH 8.0; 0.005% thimerosal) and re-equilibrated in PBS and stored at 4° C.

Aliquots of pMB1750-2360, pMB1750-2360 (Gerbu) or pPB1750-2360 IgY polyclonal antibody preparations (PEG preps) were affinity purified on the above column as follows. The column was attached to an UV monitor (ISCO), and washed with PBS. Forty ml aliquots of 2X PEG preps (filter sterilized using a 0.45μ filter and quantified by OD₂₈₀ before chromatography) was applied. The column was washed with PBS until the baseline was re-established (the column flow-through was saved), washed with BBSTween to elute nonspecifically binding antibodies and re-equilibrated with PBS. Bound antibody was eluted from the column in 4M guanidine-HCl (in 10 mM Tris-HCL, pH 8.0, 0.005% thimerosal) and the entire elution peak collected in a 15 ml tube (Falcon). The column was re-equilibrated, and the column eluate re-chromatographed as described above. The antibody preparations were quantified by UV absorbance (the elution buffer was used to zero the spectrophotometer). Approximately 10 fold higher concentrations of total purified antibody was obtained upon elution of the first chromatography pass relative to the second pass. The low yield from the second chromatography pass indicated that most of the specific antibodies were removed by the first round of chromatography.

Pools of affinity purified specific antibodies were prepared by dialysis of the column elutes after the first column chromatography pass for the pMB1750-2360, pMB 1750-2360 (Gerbu) or pPB 1750-2360 IgY polyclonal antibody preparations. The elutes were collected on ice and immediately dialyzed against a 100-fold volume of PBS at 4° C. for 2 hrs. The samples were then dialyzed against 3 changes of a 65-fold volume of PBS at 4° C. Dialysis was performed for a minimum of 8 hrs per change of PBS. The dialyzed samples were collected, centrifuged to remove insoluble debris, quantified by OD₂₈₀, and stored at 4° C.

The percentage of toxin B repeat-specific antibodies present in each preparation was determined using the quantifications of antibody yields from the first column pass (amount of specific antibody recovered after first pass/total protein loaded). The yield of repeat-specific affinity purified antibody (expressed as the percent of total protein in the preparation) in: 1) the pMB1750-2360 PEG prep was approximately 0.5%, 2) the pMB1750-2360 (Gerbu) prep was approximately 2.3%, and 3) the pPB1750-2360 prep was approximately 0.4%. Purification of a CTB IgY polyclonal antibody preparation on the same column demonstrated that the concentration of toxin B repeat specific antibodies in the CTB preparation was 0.35%.

These results demonstrate that 1) the use of Gerbu adjuvant enhanced the titer of specific antibody produced against the pMB1750-2360 protein 5-fold relative to immunization using Freunds adjuvant, and 2) the differences seen in the in vivo neutralization ability of the pMB 1750-2360 (not neutralizing) and pPB 1750-2360 (neutralizing) and CTB (neutralizing) PEG preps seen in Example 19 was not due to differences in the titers of repeat-specific antibodies in the three preparations because the titer of repeat-specific antibody was similar for all three preps; therefore the differing ability of the three antibody preparations to neutralize toxin B must reflect qualitative differences in the induced toxin B repeat-specific antibodies. To confirm that qualitative differences exist between antibodies raised in hens immunized with different recombinant proteins and/or different adjuvants, the same amount of affinity purified anti-toxin B repeat (aa 1870-2360 of toxin B) antibodies from the different preparations was administered to hamsters using the in vivo hamster model as described below.

b) In vivo Neutralization Of Toxin B Using Affinity Purified Antibody

The in vivo hamster model was utilized to assess the neutralizing ability of the affinity purified antibodies raised against recombinant toxin B proteins purified in (a) above. As well, a 4X IgY PEG preparation from a second independent immunization utilizing the pPB1750-2360 antigen with Freunds adjuvant was tested for in vivo neutralization. The results are shown in Table 31.

The results shown in Table 31 demonstrate that:

1) as shown in Example 19 and reproduced here, 1.5 mg of affinity purified antibody from pMB1750-2360 immunized hens using Freunds adjuvant does not neutralize toxin B in vivo. However, 300 μg of affinity purified antibody from similarly immunized hens utilizing Gerbu adjuvant demonstrated complete neutralization of toxin B in vivo. This demonstrates that Gerbu adjuvant, in addition to enhancing the titer of antibodies reactive to the pMB1750-2360 antigen relative to Freunds adjuvant (demonstrated in (a) above), also enhances the yield of neutralizing antibodies to this antigen, greater than 5 fold.

2) Complete in vivo neutralization of toxin B was observed with 1.5 mg of affinity purified antibody from hens immunized with pPB1750-2360

                  TABLE 31                                                         ______________________________________                                         In vivo Neutralization Of Toxin B Using Affinity Purified Antibodies                            Number Animals                                                                             Number Animals                                    Treatment Group.sup.a                                                                           Alive.sup.b Dead.sup.b                                        ______________________________________                                         Preimmune.sup.i  0           5                                                 CTB (300 μg).sup.2                                                                           5           0                                                 CTB (100 μg).sup.2                                                                           1           4                                                 pMB1750-2360 (G) (5 mg).sup.2                                                                   5           0                                                 pMB1750-2360 (G) (1.5 mg).sup.2                                                                 5           0                                                 pMB1750-2360 (G) (300 μg).sup.2                                                              5           0                                                 pMB1750-2360 (F) (1.5 mg).sup.2                                                                 0           5                                                 pPB1750-2360 (F) (1.5 mg).sup.2                                                                 5           0                                                 pPB1750-2360 (F) (300 μg).sup.2                                                              1           4                                                 CTB (100 μg).sup.3                                                                           2           3                                                 pPB1750-2360 (F) (500 μg).sup.1                                                              5           0                                                 ______________________________________                                          .sup.a C. difficile toxin B (CTB) (Tech Lab) at lethal concentration to        hamsters (25 μg) was added to the antibody (amount of specific antibod      is indicated) and incubated for one hour at 37° C.. After               incubation, this mixture was injected IP into hamsters (1/5 total mix          injected per hamster). Each treatment group received toxin premixed with       antibody raised against the indicated protein (G = gerbu adjuvant, F =         Freunds adjuvant). .sup.1 indicates the antibody was a 4X IgY PEG prep;        .sup.2 indicates the antibody was affinity purified on a pPB18502360           resin; and .sup.3 indicates that the antibody was a 1X IgY PEG prep.           .sup.b The numbers in each group represent numbers of hamsters dead or         alive, 2 hrs post IP administration of toxin/antibody mixture.           

antigen, but not with pMB1750-2360 antigen, when Freunds adjuvant was used. This demonstrates, using standardized toxin B repeat-specific antibody concentrations, that neutralizing antibodies were induced when pPB 1750-2360 but not pMB1750-2360 was used as the antigen with Freunds adjuvant.

3) Complete in vivo neutralization was observed with 300 μg of pMB1750-2360 (Gerbu) antibody, but not with 300 μg of pPB1750-2360 (Freunds) antibody. Thus the pMB1750-2360 (Gerbu) antibody has a higher titer of neutralizing antibodies than the pPB1750-2360 (Freunds) antibody.

4) Complete neutralization of toxin B was observed using 300 μg of CTB antibody affinity purified (AP)! but not 100 μg CTB antibody (AP or PEG prep). This demonstrates that greater than 100 μg of toxin B repeat-specific antibody (anti-CTB) is necessary to neutralize 25 μg toxin B in vivo in this assay, and that affinity purified antibodies specific to the toxin B repeat interval neutralize toxin B as effectively as the PEP prep of IgY raised against the entire CTB protein (shown in this assay).

5) As was observed with the initial pPB1750-2360 (IgY) PEG preparation (Example 19), complete neutralization was observed with a IgY PEG preparation isolated from a second independent group of pPB1750-2360 (Freunds) immunized hens. This demonstrates that neutralizing antibodies are reproducibly produced when hens are immunized with pPB1750-2360 protein utilizing Freund's adjuvant.

EXAMPLE 21 Diagnostic Enzyme Immunoassays For C. difficile Toxins A And B

The ability of the recombinant toxin proteins and antibodies raised against these recombinant proteins (described in the above examples) to form the basis of diagnostic assays for the detection of clostridial toxin in a sample was examined. Two immunoassay formats were tested to quantitatively detect C. difficile toxin A and toxin B from a biological specimen. The first format involved a competitive assay in which a fixed amount of recombinant toxin A or B was immobilized on a solid support (e.g., microtiter plate wells) followed by the addition of a toxin-containing biological specimen mixed with affinity-purified or PEG fractionated antibodies against recombinant toxin A or B. If toxin is present in a specimen, this toxin will compete with the immobilized recombinant toxin protein for binding to the anti-recombinant antibody thereby reducing the signal obtained following the addition of a reporter reagent. The reporter reagent detects the presence of antibody bound to the immobilized toxin protein.

In the second format, a sandwich immunoassay was developed using affinity-purified antibodies to recombinant toxin A and B. The affinity-purified antibodies to recombinant toxin A and B were used to coat microtiter wells instead of the recombinant polypeptides (as was done in the competitive assay format). Biological samples containing toxin A or B were then added to the wells followed by the addition of a reporter reagent to detect the presence of bound toxin in the well.

a) Competitive Immunoassay For The Detection Of C. difficile Toxin

Recombinant toxin A or B was attached to a solid support by coating 96 well microtiter plates with the toxin protein at a concentration of 1 μg/ml in PBS. The plates were incubated overnight at 2°-8° C. The following morning, the coating solutions were removed and the remaining protein binding sites on the wells were blocked by filling each well with a PBS solution containing 0.5% BSA and 0.05% Tween-20. Native C. difficile toxin A or B (Tech Lab) was diluted to 4 μg/ml in stool extracts from healthy Syrian hamsters (Sasco). The stool extracts were made by placing fecal pellets in a 15 ml centrifuge tube; PBS was added at 2 ml/pellet and the tube was vortexed to create a uniform suspension. The tube was then centrifuged at 2000 rpm for 5 min at room temperature. The supernatant was removed; this comprises the stool extract. Fifty μl of the hamster stool extract was pipetted into each well of the microtiter plates to serve as the diluent for serial dilutions of the 4 μg/ml toxin samples. One hundred μl of the toxin samples at 4 μg/ml was pipetted into the first row of wells in the microtiter plate, and 50 μl aliquots were removed and diluted serially down the plate in duplicate. An equal volume of affinity purified anti-recombinant toxin antibodies 1 ng/well of anti-pMA1870-2680 antibody was used for the detection of toxin A; 0.5 ng/well of anti-pMB1750-2360(Gerbu) was used for the detection of toxin B! were added to appropriate wells, and the plates were incubated at room temperature for 2 hours with gentle agitation. Wells serving as negative control contained antibody but no native toxin to compete for binding.

Unbound toxin and antibody were removed by washing the plates 3 to 5 times with PBS containing 0.05% Tween-20. Following the wash step, 100 μl of rabbit anti-chicken IgG antibody conjugated to alkaline phosphatase (Sigma) was added to each well and the plates were incubated for 2 hours at room temperature. The plates were then washed as before to remove unbound secondary antibody. Freshly prepared alkaline phosphatase substrate (1 mg/ml p-nitrophenyl phosphate (Sigma) in 50 mM Na₂ CO₃, pH 9.5; 10 mM MgCl₂) was added to each well. Once sufficient color developed, the plates were read on a Dynatech MR700 microtiter plate reader using a 410 nm filter.

The results are summarized in Tables 32 and 33. For the results shown in Table 32, the wells were coated with recombinant toxin A protein (pMA1870-2680). The amount of native toxin A added (present as an addition to solubilized hamster stool) to a given well is indicated (0 to 200 ng). Antibody raised against the recombinant toxin A protein, pMA1870-2680, was affinity purified on the an affinity column containing pPA1870-2680 (described in Example 20). As shown in Table 32, the recombinant toxin A protein and affinity-purified antitoxin can be used for the basis of a competitive immunoassay for the detection of toxin A in biological samples.

Similar results were obtained using the recombinant toxin B, pPB1750-2360, and antibodies raised against pMB1750-2360(Gerbu). For the results shown in Table 33, the wells were coated with recombinant toxin B protein (pPB1750-2360). The amount of native toxin B added (present as an addition to solubilized hamster stool) to a given well is indicated (0 to 200 ng). Antibody raised against the recombinant toxin B protein, pMB1750-2360(Gerbu), was affinity purified on the an affinity column containing pPB1850-2360 (described in Example 20). As shown in Table 33, the recombinant toxin B protein and affinity-purified antitoxin can be used for the basis of a competitive immunoassay for the detection of toxin B in biological samples.

In this competition assay, the reduction is considered significant over the background levels at all points; therefore the assay can be used to detect samples containing less than 12.5 ng toxin A/well and as little as 50-100 ng toxin B/well.

                  TABLE 32                                                         ______________________________________                                         Competitive Inhibition Of Anti-C. difficile Toxin A By Native Toxin A          ng Toxin A/Well                                                                               OD.sub.410 Readout                                              ______________________________________                                         200            0.176                                                           100            0.253                                                           50             0.240                                                           25             0.259                                                           12.5           0.309                                                           6.25           0.367                                                           3.125          0.417                                                           0              0.590                                                           ______________________________________                                    

                  TABLE 33                                                         ______________________________________                                         Competitive Inhibition Of Anti-C. difficile Toxin B By Native Toxin B          ng Toxin A/Well                                                                               OD.sub.410 Readout                                              ______________________________________                                         200            0.392                                                           100            0.566                                                           50             0.607                                                           25             0.778                                                           12.5           0.970                                                           6.25           0.902                                                           3.125          1.040                                                           0              1.055                                                           ______________________________________                                    

These competitive inhibition assays demonstrate that native C. difficile toxins and recombinant C. difficile toxin proteins can compete for binding to antibodies raised against recombinant C. difficile toxins demonstrating that these anti-recombinant toxin antibodies provide effective diagnostic reagents.

b) Sandwich Immunoassay For The Detection Of C. difficile Toxin

Affinity-purified antibodies against recombinant toxin A or toxin B were immobilized to 96 well microtiter plates as follows. The wells were passively coated overnight at 4° C. with affinity purified antibodies raised against either pMA1870-2680 (toxin A) or pMB1750-2360(Gerbu) (toxin B). The antibodies were affinity purified as described in Example 20. The antibodies were used at a concentration of 1 μg/ml and 100 μl was added to each microtiter well. The wells were then blocked with 200 μl of 0.5% BSA in PBS for 2 hours at room temperature and the blocking solution was then decanted. Stool samples from healthy Syrian hamsters were resuspended in PBS, pH 7.4 (2 ml PBS/stool pellet was used to resuspend the pellets and the sample was centrifuged as described above). The stool suspension was then spiked with native C. difficile toxin A or B (Tech Lab) at 4 μg/ml. The stool suspensions containing toxin (either toxin A or toxin B) were then serially diluted two-fold in stool suspension without toxin and 50 μl was added in duplicate to the coated microtiter wells. Wells containing stool suspension without toxin served as the negative control.

The plates were incubated for 2 hours at room temperature and then were washed three times with PBS. One hundred μl of either goat anti-native toxin A or goat anti-native toxin B (Tech Lab) diluted 1:1000 in PBS containing 1% BSA and 0.05% Tween 20 was added to each well. The plates were incubated for another 2 hours at room temperature. The plates were then washed as before and 100 μl of alkaline phosphatase-conjugated rabbit anti-goat IgG (Cappel, Durham, N.C.) was added at a dilution of 1:1000. The plates were incubated for another 2 hours at room temperature. The plates were washed as before then developed by the addition of 100 μl well of a substrate solution containing 1 mg/ml p-nitrophenyl phosphate (Sigma) in 50 mM Na₂ CO₃, pH 9.5; 10 mM MgCl₂. The absorbance of each well was measured using a plate reader (Dynatech) at 410 nm. The assay results are shown in Tables 34 and 35.

                  TABLE 34                                                         ______________________________________                                         C. difficile Toxin A Detection In Stool                                        Using Affinity-Purified Antibodies Against Toxin A                             ng Toxin A/Welll                                                                              OD.sub.410 Readout                                              ______________________________________                                         200            0.9                                                             100            0.8                                                             50             0.73                                                            25             0.71                                                            12.5           0.59                                                            6.25           0.421                                                           0              0                                                               ______________________________________                                    

                  TABLE 35                                                         ______________________________________                                         C. difficile Toxin B Detection In Stool                                        Using Affinity-Purified Antibodies Against Toxin B                             ng Toxin B/Well                                                                               OD.sub.410 Readout                                              ______________________________________                                         200            1.2                                                             100            0.973                                                           50             0.887                                                           25             0.846                                                           12.5           0.651                                                           6.25           0.431                                                           0              0.004                                                           ______________________________________                                    

The results shown in Tables 34 and 35 show that antibodies raised against recombinant toxin A and toxin B fragments can be used to detect the presence of C. difficile toxin in stool samples. These antibodies form the basis for a sensitive sandwich immunoassay which is capable of detecting as little as 6.25 ng of either toxin A or B in a 50 μl stool sample. As shown above in Tables 34 and 35, the background for this sandwich immunoassay is extremely low; therefore, the sensitivity of this assay is much lower than 6.25 ng toxin/well. It is likely that toxin levels of 0.5 to 1.0 pg/well could be detected by this assay.

The results shown above in Tables 32-35 demonstrate clear utility of the recombinant reagents in C. difficile toxin detection systems.

EXAMPLE 22 Construction And Expression Of C. botulinum C Fragment Fusion Proteins

The C. botulinum type A neurotoxin gene has been cloned and sequenced Thompson, et al., Eur. J. Biochem. 189:73 (1990)!. The nucleotide sequence of the toxin gene is available from the EMBL/GenBank sequence data banks under the accession number X52066; the nucleotide sequence of the coding region is listed in SEQ ID NO:27. The amino acid sequence of the C. botulinum type A neurotoxin is listed in SEQ ID NO:28. The type A neurotoxin gene is synthesized as a single polypeptide chain which is processed to form a dimer composed of a light and a heavy chain linked via disulfide bonds. The 50 kD carboxy-terminal portion of the heavy chain is referred to as the C fragment or the H_(c) domain.

Previous attempts by others to express polypeptides comprising the C fragment of C. botulinum type A toxin as a native polypeptide (e.g., not as a fusion protein) in E. coli have been unsuccessful H. F. LaPenotiere, et al. in Botulinum and Tetanus Neurotoxins, DasGupta, Ed., Plenum Press, New York (1993), pp. 463-466!. Expression of the C fragment as a fusion with the E. coli MBP was reported to result in the production of insoluble protein (H. F. LaPenotiere, et al., supra).

In order to produce soluble recombinant C fragment proteins in E. coli, fusion proteins comprising a synthetic C fragment gene derived from the C. botulinum type A toxin and either a portion of the C. difficile toxin protein or the MBP were constructed. This example involved a) the construction of plasmids encoding C fragment fusion proteins and b) expression of C. botulinum C fragment fusion proteins in E. coli.

a) Construction Of Plasmids Encoding C Fragment Fusion Proteins

In Example 11, it was demonstrated that the C. difficile toxin A repeat domain can be efficiently expressed and purified in E. coli as either native (expressed in the pET 23a vector in clone pPA1870-2680) or fusion (expressed in the pMALc vector as a fusion with the E. coli MBP in clone pMA1870-2680) proteins. Fusion proteins comprising a fusion between the MBP, portions of the C. difficile toxin A repeat domain (shown to be expressed as a soluble fusion protein) and the C fragment of the C. botulinum type A toxin were constructed. A fusion protein comprising the C fragment of the C. botulinum type A toxin and the MBP was also constructed.

FIG. 25 provides a schematic representation of the botulinal fusion proteins along with the donor constructs containing the C. difficile toxin A sequences or C. botulinum C fragment sequences which were used to generate the botulinal fusion proteins. In FIG. 25, the solid boxes represent C. difficile toxin A gene sequences, the open boxes represent C. botulinum C fragment sequences and the solid black ovals represent the E. coli MBP. When the name for a restriction enzyme appears inside parenthesis, this indicates that the restriction site was destroyed during construction. An asterisk appearing with the name for a restriction enzyme indicates that this restriction site was recreated at the cloning junction.

In FIG. 25, a restriction map of the pMA1870-2680 and pPA1100-2680 constructs (described in Example 11) which contain sequences derived from the C. difficile toxin A repeat domain are shown; these constructs were used as the source of C. difficile toxin A gene sequences for the construction of plasmids encoding fusions between the C. botulinum C fragment gene and the C. difficile toxin A gene. The pMA1870-2680 expression construct expresses high levels of soluble, intact fusion protein (20 mg/liter culture) which can be affinity purified on an amylose column (purification described in Example 11d).

The pAlterBot construct (FIG. 25) was used as the source of C. botulinum C fragment gene sequences for the botulinal fusion proteins. pAlterBot was obtained from J. Middlebrook and R. Lemley at the U.S. Department of Defense. pAlterBot contains a synthetic C. botulinum C fragment inserted in to the pALTER-1® vector (Promega). This synthetic C fragment gene encodes the same amino acids as does the naturally occurring C fragment gene. The naturally occurring C fragment sequences, like most clostridial genes, are extremely A/T rich (Thompson et al., supra). This high A/T content creates expression difficulties in E. coli and yeast due to altered codon usage frequency and fortuitous polyadenylation sites, respectively. In order to improve the expression of C fragment proteins in E. coli, a synthetic version of the gene was created in which the non-preferred codons were replaced with preferred codons.

The nucleotide sequence of the C. botulinum C fragment gene sequences contained within pAlterBot is listed in SEQ ID NO:22. The first six nucleotides (ATGGCT) encode a methionine and alanine residue, respectively. These two amino acids result from the insertion of the C. botulinum C fragment sequences into the pALTER® vector and provide the initiator methionine residue. The amino acid sequence of the C. botulinum C fragment encoded by the sequences contained within pAlterBot is listed in SEQ ID NO:23. The first two amino acids (Met Ala) are encoded by vector-derived sequences. From the third amino acid residue onward (Arg), the amino acid sequence is identical to that found in the C. botulinum type A toxin gene.

The pMA1870-2680, pPA1100-2680 and pAlterBot constructs were used as progenitor plasmids to make expression constructs in which fragments of the C. difficile toxin A repeat domain were expressed as genetic fusions with the C. botulinum C fragment gene using the pMAL-c expression vector (New England BioLabs). The pMAL-c expression vector generates fusion proteins which contain the MBP at the amino-terminal end of the protein. A construct, pMBot, in which the C. botulinum C fragment gene was expressed as a fusion with only the MBP was constructed (FIG. 25). Fusion protein expression was induced from E. coli strains harboring the above plasmids, and induced protein was affinity purified on an amylose resin column.

i) Construction Of pBlueBot

In order to facilitate the cloning of the C. botulinum C fragment gene sequences into a number of desired constructs, the botulinal gene sequences were removed from pAlterBot and were inserted into the pBluescript plasmid (Stratagene) to generate pBlueBot (FIG. 25). pBlueBot was constructed as follows. Bacteria containing the pAlterBot plasmid were grown in medium containing tetracycline and plasmid DNA was isolated using the QIAprep-spin Plasmid Kit (Qiagen). One microgram of pAlterBot DNA was digested with NcoI and the resulting 3' recessed sticky end was made blunt using the Klenow fragment of DNA polymerase I (here after the Klenow fragment). The pAlterBot DNA was then digested with HindIII to release the botulinal gene sequences (the Bot insert) as a blunt (filled NcoI site)-HindIII fragment. pBluescript vector DNA was prepared by digesting 200 ng of pBluescript DNA with SmaI and HindIII. The digestion products from both plasmids were resolved on an agarose gel. The appropriate fragments were removed from the gel, mixed and purified utilizing the Prep-a-Gene kit (BioRad). The eluted DNA was then ligated using T4 DNA ligase and used to transform competent DH5α cells (Gibco-BRL). Host cells were made competent for transformation using the calcium chloride protocol of Sambrook et al., supra at 1.82-1.83. Recombinant clones were isolated and confirmed by restriction digestion using standard recombinant molecular biology techniques (Sambrook et al, supra). The resultant clone, pBlueBot, contains several useful unique restriction sites flanking the Bot insert (i.e., the C. botulinum C fragment sequences derived from pAlterBot) as shown in FIG. 25.

ii) Construction Of C. difficile/C. botulinum /MBP Fusion Proteins

Constructs encoding fusions between the C. difficile toxin A gene and the C. botulinum C fragment gene and the MBP were made utilizing the same recombinant DNA methodology outlined above; these fusion proteins contained varying amounts of the C. difficile toxin A repeat domain.

The pMABot clone contains a 2.4 kb insert derived from the C. difficile toxin A gene fused to the Bot insert (i.e., the C. botulinum C fragment sequences derived from pAlterBot). pMABot (FIG. 25) was constructed by mixing gel-purified DNA from NotI/HindIII digested pBlueBot (the 1.2 kb Bot fragment), SpeI/NotI digested pPA1100-2680 (the 2.4 kb C. difficile toxin A repeat fragment) and XbaI/HindIII digested pMAL-c vector. Recombinant clones were isolated, confirmed by restriction digestion and purified using the QIAprep-spin Plasmid Kit (Qiagen). This clone expresses the toxin A repeats and the botulinal C fragment protein sequences as an in-frame fusion with the MBP.

The pMCABot construct contains a 1.0 kb insert derived from the C. difficile toxin A gene fused to the Bot insert (i.e, the C. botulinum C fragment sequences derived from pAlterBot). pMCABot was constructed by digesting the pMABot clone with EcoRI to remove the 5' end of the C. difficile toxin A repeat (see FIG. 25, the pMAL-c vector contains a EcoRI site 5' to the C. difficile insert in the pMABot clone). The restriction sites were filled and religated together after gel purification. The resultant clone (pMCABot, FIG. 25) generated an in-frame fusion between the MBP and the remaining 3' portion of the C. difficile toxin A repeat domain fused to the Bot gene.

The pMNABot clone contains the 1 kb SpeI/EcoRI (filled) fragment from the C. difficile toxin A repeat domain (derived from clone pPA1100-2680) and the 1.2 kb C. botulinum C fragment gene as a NcoI (filled)/HindIII fragment (derived from pAlterBot). These two fragments were inserted into the pMAL-c vector digested with XbaI/HindIII. The two insert fragments were generated by digestion of the appropriate plasmid with EcoRI (pPA1100-2680) or NcoI (pAlterBot) followed by treatment with the Klenow fragment. After treatment with the Klenow fragment, the plasmids were digested with the second enzyme (either SpeI or HindIII). All three fragments were gel purified, mixed and Prep-a-Gene purified prior to ligation. Following ligation and transformation, putative recombinants were analyzed by restriction analysis; the EcoRI site was found to be regenerated at the fusion junction, as was predicted for a fusion between the filled EcoRI and NcoI sites.

A construct encoding a fusion protein between the botulinal C fragment gene and the MBP gene was constructed (i.e., this fusion lacks any C. difficile toxin A gene sequences) and termed pMBot. The pMBot construct was made by removal of the C. difficile toxin A sequences from the pMABot construct and fusing the C fragment gene sequences to the MBP. This was accomplished by digestion of pMABot DNA with StuI (located in the pMALc polylinker 5' to the XbaI site) and XbaI (located 3' to the NotI site at the toxA-Bot fusion junction), filling in the XbaI site using the Klenow fragment, gel purifying the desired restriction fragment, and ligating the blunt ends to circularize the plasmid. Following ligation and transformation, putative recombinants were analyzed by restriction mapping of the Bot insert (i.e, the C. botulinum C fragment sequences).

b) Expression Of C. botulinum C Fragment Fusion Proteins In E. coli

Large scale (1 liter) cultures of the pMAL-c vector, and each recombinant construct described above in (a) were grown, induced, and soluble protein fractions were isolated as described in Example 18. The soluble protein extracts were chromatographed on amylose affinity columns to isolate recombinant fusion protein. The purified recombinant fusion proteins were analyzed by running samples on SDS-PAGE gels followed by Coomassie staining and by Western blot analysis as described Williams et al, (1994) supra!. In brief, extracts were prepared and chromatographed in column buffer (10 mM NaPO₄, 0.5M NaCl, 10 mM β-mercaptoethanol, pH 7.2) over an amylose resin (New England Biolabs) column, and eluted with column buffer containing 10 mM maltose as described Williams, et al. (1994), supra!. An SDS-PAGE gel containing the purified protein samples stained with Coomassie blue is shown in FIG. 26.

In FIG. 26, the following samples were loaded. Lanes 1-6 contain protein purified from E. coli containing the pMAL-c, pPA1870-2680, pMABot, pMNABot, pMCABot and pMBot plasmids, respectively. Lane 7 contains broad range molecular weight protein markers (BioRad).

The protein samples were prepared for electrophoresis by mixing 5 μl of eluted protein with 5 μl of 2X SDS-PAGE sample buffer (0.125 mM Tris-HCl, pH 6.8, 2 mM EDTA, 6% SDS, 20% glycerol, 0.025% bromophenol blue; β-mercaptoethanol is added to 5% before use). The samples were heated to 95° C. for 5 min, then cooled and loaded on a 7.5% agarose SDS-PAGE gel. Broad range molecular weight protein markers were also loaded to allow estimation of the MW of identified fusion proteins. After electrophoresis, protein was detected generally by staining the gel with Coomassie blue.

In all cases the yields were in excess of 20 mg fusion protein per liter culture (see Table 36) and, with the exception of the pMCABot protein, a high percentage (i.e., greater than 20-50% of total eluted protein) of the eluted fusion protein was of a MW predicted for the full length fusion protein (FIG. 26). It was estimated (by visual inspection) that less than 10% of the pMCABot fusion protein was expressed as the full length fusion protein.

                  TABLE 36                                                         ______________________________________                                         Yield Of Affinity Purified C. botulinum C Fragment/MBP Fusion Proteins                     Yield (mg/liter                                                                            Percentage Of Total                                    Construct   of Culture) Soluble Protein                                        ______________________________________                                         pMABot      24          5.0                                                    pMCABot     34          5.0                                                    pMNABot     40          5.5                                                    pMBot       22          5.0                                                    pMA1870-2680                                                                               40          4.8                                                    ______________________________________                                    

These results demonstrate that high level expression of intact C. botulinum C fragment/C. difficile toxin A fusion proteins in E. coli is feasible using the pMAL-c expression system. These results are in contrast to those reported by H. F. LaPenotiere, et al. (1993), supra. In addition, these results show that it is not necessary to fuse the botulinal C fragment gene to the C. difficile toxin A gene in order to produce a soluble fusion protein using the pMAL-c system in E. coli.

In order to determine whether the above-described botulinal fusion proteins were recognized by anti-C. botulinum toxin A antibodies, Western blots were performed. Samples containing affinity-purified proteins from E. coli containing the pMABot, pMCABot, pMNABot, pMBot, pMA1870-2680 or pMALc plasmids were analyzed. SDS-PAGE gels (7.5% acrylamide) were loaded with protein samples purified from each expression construct. After electrophoresis, the gels were blotted and protein transfer was confirmed by Ponceau S staining (as described in Example 12b).

Following protein transfer, the blots were blocked by incubation for 1 hr at 20° C. in blocking buffer PBST (PBS containing 0.1% Tween 20 and 5% dry milk)!. The blots were then incubated in 10 ml of a solution containing the primary antibody; this solution comprised a 1/500 dilution of an anti-C. botulinum toxin A IgY PEG prep (described in Example 3) in blocking buffer. The blots were incubated for 1 hr at room temperature in the presence of the primary antibody. The blots were washed and developed using a rabbit anti-chicken alkaline phosphatase conjugate (Boehringer Mannheim) as the secondary antibody as follows. The rabbit anti-chicken antibody was diluted to 1 μg/ml in blocking buffer (10 ml final volume per blot) and the blots were incubated at room temperature for 1 hour in the presence of the secondary antibody. The blots were then washed successively with PBST, BBS-Tween and 50 mM Na₂ CO₃, pH 9.5. The blots were then developed in freshly-prepared alkaline phosphatase substrate buffer (100 μg/ml nitro blue tetrazolium, 50 μg/ml 5-bromo-chloro-indolylphosphate, 5 mM MgCl₂ in 50 mM Na₂ CO₃, pH 9.5). Development was stopped by flooding the blots with distilled water and the blots were air dried.

This Western blot analysis detected anti-C. botulinum toxin reactive proteins in the pMABot, pMCABot, pMNABot and pMBot protein samples (corresponding to the predicted full length proteins identified above by Coomassie staining in FIG. 26), but not in the pMA1100-2680 or pMALc protein samples.

These results demonstrate that the relevant fusion proteins purified on an amylose resin as described above in section a) contained immunoreactive C. botulinum C fragment protein as predicted.

EXAMPLE 23 Generation Of Neutralizing Antibodies By Nasal Administration Of pMBot Protein

The ability of the recombinant botulinal toxin proteins produced in Example 22 to stimulate a systemic immune response against botulinal toxin epitopes was assessed. This example involved: a) the evaluation of the induction of serum IgG titers produced by nasal or oral administration of botulinal toxin-containing C. difficile toxin A fusion proteins and b) the in vivo neutralization of C. botulinum type A neurotoxin by anti- recombinant C. botulinum C fragment antibodies.

a) Evaluation Of The Induction Of Serum IgG Titers Produced By Nasal Or Oral Administration Of Botulinal Toxin-Containing C. difficile Toxin A Fusion Proteins

Six groups containing five 6 week old CF female rats (Charles River) per group were immunized nasally or orally with one of the following three combinations using protein prepared in Example 22: (1) 250 μg pMBot protein per rat (nasal and oral); 2) 250 lag pMABot protein per rat (nasal and oral); 3) 125 μg pMBot admixed with 125 μg pMA1870-2680 per rat (nasal and oral). A second set of 5 groups containing 3 CF female rats/group were immunized nasally or orally with one of the following combinations (4) 250 μg pMNABot protein per rat (nasal and oral) or 5) 250 μg pMAL-c protein per rat (nasal and oral).

The fusion proteins were prepared for immunization as follows. The proteins (in column buffer containing 10 mM maltose) were diluted in 0.1M carbonate buffer, pH 9.5 and administered orally or nasally in a 200 μl volume. The rats were lightly sedated with ether prior to administration. The oral dosing was accomplished using a 20 gauge feeding needle. The nasal dosing was performed using a P-200 micro-pipettor (Gilson). The rats were boosted 14 days after the primary immunization using the techniques described above and were bled 7 days later. Rats from each group were lightly etherized and bled from the tail. The blood was allowed to clot at 37° C. for 1 hr and the serum was collected.

The serum from individual rats was analyzed using an ELISA to determine the anti-C. botulinum type A toxin IgG serum titer. The ELISA protocol used is a modification of that described in Example 13c. Briefly, 96-well microtiter plates (Falcon, Pro-Bind Assay Plates) were coated with C. botulinum type A toxoid (prepared as described in Example 3a) by placing 100 μl volumes of C. botulinum type A toxoid at 2.5 μg/ml in PBS containing 0.005% thimerosal in each well and incubating overnight at 4° C. The next morning, the coating suspensions were decanted and all wells were washed three times using PBS.

In order to block non-specific binding sites, 100 μl of blocking solution 0.5% BSA in PBS! was then added to each well and the plates were incubated for 1 hr at 37° C. The blocking solution was decanted and duplicate samples of 150 μl of diluted rat serum added to the first well of a dilution series. The initial testing serum dilution was 1:30 in blocking solution containing 0.5% Tween 20 followed by 5-fold dilutions into this solution. This was accomplished by serially transferring 30 μl aliquots to 120 μl blocking solution containing 0.5% Tween 20, mixing, and repeating the dilution into a fresh well. After the final dilution, 30 μl was removed from the well such that all wells contained 120 μl final volume. A total of 3 such dilutions were performed (4 wells total). The plates were incubated 1 hr at 37° C. Following this incubation, the serially diluted samples were decanted and the wells were washed six times using PBS containing 0.5% Tween 20 (PBST). To each well, 100 μl of a rabbit anti-Rat IgG alkaline phosphatase (Sigma) diluted (1/1000) in blocking buffer containing 0.5% Tween 20 was added and the plate was incubated for 1 hr at 37° C. The conjugate solutions were decanted and the plates were washed as described above, substituting 50 mM Na₂ CO₃, pH 9.5 for the PBST in the final wash. The plates were developed by the addition of 100 μl of a solution containing 1 mg/ml para-nitro phenyl phosphate (Sigma) dissolved in 50 mM Na₂ CO₃, 10 mM MgCl₂, pH 9.5 to each well, and incubating the plates at room temperature in the dark for 5-45 min. The absorbency of each well was measured at 410 nm using a Dynatech MR 700 plate reader. The results are summarized in Tables 37 and 38 and represent mean serum reactivities of individual mice.

                                      TABLE 37                                     __________________________________________________________________________     Determination Of Anti-C. botulinum Type A Toxin Serum IgG Titers               Following Immunization With C. botulinum C Fragment-Containing Fusion          Proteins                                                                                  Nasal          Oral                                                 Route of Immunization                                                                         pMBot &        pMBot&                                                 PRE-     pMA1870-             pMA1870-                                   Immunogen                                                                            IMMUNE                                                                              pMBot                                                                              2680  pMABot                                                                              pMBot                                                                              2680  pMABot                                     __________________________________________________________________________     Dilution                                                                       1:30  0.080                                                                               1.040                                                                              1.030 0.060                                                                               0.190                                                                              0.080 0.120                                      1:150 0.017                                                                               0.580                                                                              0.540 0.022                                                                               0.070                                                                              0.020 0.027                                      1:750 0.009                                                                               0.280                                                                              0.260 0.010                                                                               0.020                                                                              0.010 0.014                                      1:3750                                                                               0.007                                                                               0.084                                                                              0.090 0.009                                                                               0.009                                                                              0.010 0.007                                      # Rats     5   5     5    5   2     2                                          Tested                                                                         __________________________________________________________________________      *Numbers represent the average values obtained from two ELISA plates,          standardized utilizing the preimmune control.                            

                                      TABLE 38                                     __________________________________________________________________________     Determination Of Anti-C. botulinum Type A Toxin Serum IgG Titers               Following Immunization With C. botulinum                                       C Fragment-Containing Fusion Proteins                                                 PRE-                                                                    Immunogen                                                                             IMMUNE                                                                              pMBot                                                                              pMABot                                                                              pMNABot                                                                             pMNABot                                              __________________________________________________________________________     Dilution                                                                       1:30   0.040                                                                               0.557                                                                              0.010                                                                               0.015                                                                               0.010                                                1:150  0.009                                                                               0.383                                                                              0.001                                                                               0.003                                                                               0.002                                                1:750  0.001                                                                               0.140                                                                              0.000                                                                               0.000                                                                               0.000                                                1:3750 0.000                                                                               0.040                                                                              0.000                                                                               0.000                                                                               0.000                                                # Rats Tested                                                                              1   1    3    3                                                    __________________________________________________________________________

The above ELISA results demonstrate that reactivity against the botulinal fusion proteins was strongest when the route of administration was nasal; only weak responses were stimulated when the botulinal fusion proteins were given orally. Nasally delivered pMbot and pMBot admixed with pMA1870-2680 invoked the greatest serum IgG response. These results show that only the pMBot protein is necessary to induce this response, since the addition of the pMA1870-2680 protein did not enhance antibody response (Table 37). Placement of the C. difficile toxin A fragment between the MBP and the C. botulinum C fragment protein dramatically reduced anti-bot IgG titer (see results using pMABot, pMCABot and pMNABot proteins).

This study demonstrates that the pMBot protein induces a strong serum IgG response directed against C. botulinum type A toxin when nasally administered.

b) In Vivo Neutralization Of C. botulinum Type A Neurotoxin By Anti- Recombinant C. botulinum C Fragment Antibodies

The ability of the anti-C. botulinum type A toxin antibodies generated by nasal administration of recombinant botulinal fusion proteins in rats (Example 22) to neutralize C. botulinum type A toxin was tested in a mouse neutralization model. The mouse model is the art accepted method for detection of botulinal toxins in body fluids and for the evaluation of anti-botulinal antibodies E. J. Schantz and D. A. Kautter, J. Assoc. Off. Anal. Chem. 61:96 (1990) and Investigational New Drug (BB-IND-3703) application by the Surgeon General of the Department of the Army to the Federal Food and Drug Administration!. The anti-C. botulinum type A toxin antibodies were prepared as follows.

Rats from the group given pMBot protein by nasal administration were boosted a second time with 250 μg pMBot protein per rat and serum was collected 7 days later. Serum from one rat from this group and from a preimmune rat was tested for anti-C. botulinum type A toxin neutralizing activity in the mouse neutralization model described below.

The LD₅₀ of a solution of purified C. botulinum type A toxin complex, obtained from Dr. Eric Johnson (University of Wisconsin Madison), was determined using the intraperitoneal (IP) method of Schantz and Kautter J. Assoc. Off. Anal. Chem. 61:96 (1978)! using 18-22 gram female ICR mice and was found to be 3500 LD₅₀ /ml. The determination of the LD₅₀ was performed as follows. A Type A toxin standard was prepared by dissolving purified type A toxin complex in 25 mM sodium phosphate buffer, pH 6.8 to yield a stock toxin solution of 3.15×10⁷ LD₅₀ /mg. The OD₂₇₈ of the solution was determined and the concentration was adjusted to 10-20 μg/ml. The toxin solution was then diluted 1:100 in gel-phosphate (30 mM phosphate, pH 6.4; 0.2% gelatin). Further dilutions of the toxin solution were made as shown below in Table 39. Two mice were injected IP with 0.5 ml of each dilution shown and the mice were observed for symptoms of botulism for a period of 72 hours.

                  TABLE 39                                                         ______________________________________                                         Determination Of The LD.sub.50 Of Purified                                     C. botulinum Type A Toxin Complex                                              Dilution          Number Dead At 72 hr                                         ______________________________________                                         1:320             2/2                                                          1:640             2/2                                                          1:1280            2/2                                                          1:2560            0/2 (sick after 72 hr)                                       1:5120            0/2 (no symptoms)                                            ______________________________________                                    

From the results shown in Table 39, the toxin titer was assumed to be between 2560 LD₅₀ /ml and 5120 LD₅₀ /ml (or about 3840 LD₅₀ /ml). This value was rounded to 3500 LD₅₀ /ml for the sake of calculation.

The amount of neutralizing antibodies present in the serum of rats immunized nasally with pMBot protein was then determined. Serum from two rats boosted with pMBot protein as described above and preimmune serum from one rat was tested as follows. The toxin standard was diluted 1:100 in gel-phosphate to a final concentration of 350 LD₅₀ /ml. One milliliter of the diluted toxin standard was mixed with 25 μl of serum from each of the three rats and 0.2 ml of gel-phosphate. The mixtures were incubated at room temperature for 30 rain with occasional mixing. Each of two mice were injected with IP with 0.5 ml of the mixtures. The mice were observed for signs of botulism for 72 hr. Mice receiving serum from rats immunized with pMBot protein neutralized this challenge dose. Mice receiving preimmune rat serum died in less than 24 hr.

The amount of neutralizing anti-toxin antibodies present in the serum of rats immunized with pMBot protein was then quantitated. Serum antibody titrations were performed by mixing 0.1 ml of each of the antibody dilutions (see Table 40) with 0.1 ml of a 1:10 dilution of stock toxin solution (3.5×10⁴ LD₅₀ /ml) with 1.0 ml of gel-phosphate and injecting 0.5 ml IP into 2 mice per dilution. The mice were then observed for signs of botulism for 3 days (72 hr). The results are tabulated in Table 39.

As shown in Table 40 pMBot serum neutralized C. botulinum type A toxin complex when used at a dilution of 1:320 or less. A mean neutralizing value of 168 IU/ml was obtained for the pMBot serum (an IU is defined as 10,000 mouse LD₅₀). This value translates to a circulating serum titer of about 3.7 IU/mg of serum protein. This neutralizing titer is comparable to the commercially available bottled concentrated (Connaught Laboratories, Ltd.) horse anti-C. botulinum antiserum. A 10 ml vial of Connaught antiserum contains about 200 mg/ml of protein; each ml can neutralize 750

                  TABLE 40                                                         ______________________________________                                         Quantitation Of Neutralizing Antibodies In pMBot Sera                                       pMBot.sup.a                                                       Dilution       Rat 1        Rat 2                                              ______________________________________                                         1:20           2/2          2/2                                                1:40           2/2          2/2                                                1:80           2/2          2/2                                                1:160          2/2          2/2                                                1:320          2/2.sup.b    2/2.sup.b                                          1:640          0/2          0/2                                                1:1280         0/2          0/2                                                1:2560         0/2          0/2                                                ______________________________________                                          .sup.a Numbers represent the number of mice surviving at 72 hours which        received serum taken from rats immunized with the pMBot protein.               .sup.b These mice survived but were sick after 72 hr.                    

IU of C. botulinum type A toxin. After administration of one vial to a human, the circulating serum titer of the Connaught preparation would be approximately 25 IU/ml assuming an average serum volume of 3 liters). Thus, the circulating anti-C. botulinum titer seen in rats nasally immunized with pMBot protein (168 IU/ml) is 6.7 time higher than the necessary circulation titer of anti-C. botulinum antibody needed to be protective in humans.

These results demonstrate that antibodies capable of neutralizing C. botulinum type A toxin are induced when recombinant C. botulinum C fragment fusion protein produced in E. coli is used as an immunogen.

EXAMPLE 24 Production Of Soluble C. botulinum C Fragment Protein Substantially Free Of Endotoxin Contamination

Example 23 demonstrated that neutralizing antibodies are generated by immunization with the pMBot protein expressed in E. coli. These results showed that the pMBot fusion protein is a good vaccine candidate. However, immunogens suitable for use as vaccines should be pyrogen-free in addition to having the capability of inducing neutralizing antibodies. Expression clones and conditions that facilitate the production of C. botulinum C fragment protein for utilization as a vaccine were developed.

The example involved: (a) determination of pyrogen content of the pMBot protein; (b) generation of C. botulinum C fragment protein free of the MBP; (c) expression of C. botulinum C fragment protein using various expression vectors; and (d) purification of soluble C. botulinum C fragment protein substantially free of significant endotoxin contamination.

a) Determination Of The Pyrogen Content Of The pMBot Protein

In order to use a recombinant antigen as a vaccine in humans or other animals, the antigen preparation must be shown to be free of pyrogens. The most significant pyrogen present in preparations of recombinant proteins produced in gram-negative bacteria, such as E. coli, is endotoxin F. C. Pearson, Pyrogens: endotoxins, LAL testing and depyrogentaion, (1985) Marcel Dekker, New York, pp. 23-56!. To evaluate the utility of the pMBot protein as a vaccine candidate, the endotoxin content in MBP fusion proteins was determined.

The endotoxin content of recombinant protein samples was assayed utilizing the Limulus assay (LAL kit; Associates of Cape Cod) according to the manufacturer's instructions. Samples of affinity-purified pMal-c protein and pMA1870-2680 were found to contain high levels of endotoxin >50,000 EU/mg protein; EU (endotoxin unit)!. This suggested that MBP- or toxin A repeat-containing fusions with the botulinal C fragment should also contain high levels of endotoxin. Accordingly, removal of endotoxin from affinity-purified pMal-c and pMBot protein preparations was attempted as follows.

Samples of pMal-c and pMBot protein were depyrogenated with polymyxin to determine if the endotoxin could be easily removed. The following amount of protein was treated: 29 ml at 4.8 OD₂₈₀ /ml for pMal-c and 19 mls at 1.44 OD₂₈₀ /ml for pMBot. The protein samples were dialyzed extensively against PBS and mixed in a 50 ml tube (Falcon) with 0.5 ml PBS-equilibrated polymyxin B (Affi-Prep Polymyxin, BioRad). The samples were allowed to mix by rotating the tubes overnight at 4° C. The polymyxin was pelleted by centrifugation for 30 min in a bench top centrifuge at maximum speed (approximately 2000× g) and the supernatant was removed. The recovered protein (in the supernatant) was quantified by OD₂₈₀, and the endotoxin activity was assayed by LAL. In both cases only approximately 1/3 of the input protein was recovered and the polymyxin-treated protein retained significant endotoxin contamination (approximately 7000 EU/mg of pMBot).

The depyrogenation experiment was repeated using an independently purified pMal-c protein preparation and similar results were obtained. From these studies it was concluded that significant levels of endotoxin copurifies with these MBP fusion proteins using the amylose resin. Furthermore, this endotoxin cannot be easily removed by polymyxin treatment.

These results suggest that the presence of the MBP sequences on the fusion protein complicated the removal of endotoxin from preparations of the pMBot protein.

b) Generation Of C. botulinum C Fragment Protein Free Of The MBP

It was demonstrated that the pMBot fusion protein could not be easily purified from contaminating endotoxin in section a) above. The ability to produce a pyrogen-free (e.g., endotoxin-free) preparation of soluble botulinal C fragment protein free of the MBP tag was next investigated. The pMBot expression construct was designed to facilitate purification of the botulinal C fragment from the MBP tag by cleavage of the fusion protein by utilizing an engineered Factor Xa cleavage site present between the MBP and the botulinal C fragment. The Factor Xa cleavage was performed as follows.

Factor Xa (New England Biolabs) was added to the pMBot protein (using a 0.1-1.0% Factor Xa/pMBot protein ratio) in a variety of buffer conditions e.g., PBS-NaCl (PBS containing 0.5M NaCl), PBS-NaCl containing 0.2% Tween 20, PBS, PBS containing 0.2% Tween 20, PBS-C (PBS containing 2 mM CaCl₂), PBS-C containing either 0.1 or 0.5% Tween 20, PBS-C containing either 0.1 or 0.5% NP-40, PBS-C containing either 0.1 or 0.5% Triton X-100, PBS-C containing 0.1% sodium deoxycholate, PBS-C containing 0.1% SDS!. The Factor Xa digestions were incubated for 12-72 hrs at room temperature.

The extent of cleavage was assessed by Western blot or Coomassie blue staining of proteins following electrophoresis on denaturing SDS-PAGE gels, as described in Example 22. Cleavage reactions (and control samples of uncleaved pMBot protein) were centrifuged for 2 min in a microfuge to remove insoluble protein prior to loading the samples on the gel. The Factor Xa treated samples were compared with uncleaved, uncentrifuged pMBot samples on the same gel. The results of this analysis is summarized below.

1) Most (about 90%) pMBot protein could be removed by centrifugation, even when uncleaved control samples were utilized. This indicated that the pMBot fusion protein was not fully soluble (i.e., it exists as a suspension rather than as a solution). This result was consistent with the observation that most affinity-purified pMBot protein precipitates after long term storage (>2 weeks) at 4° C. Additionally, the majority (i.e., 75%) of induced pMBot protein remains in the pellet after sonication and clarification of the induced E. coli. Resuspension of these insoluble pellets in PBS followed by sonication results in partial solubilization of the insoluble pMBot protein in the pellets.!

2) The portion of pMBot protein that is fully in solution (about 10% of pMBot protein) is completely cleaved by Factor Xa, but the cleaved (released) botulinal C fragment is relatively insoluble such that only the cleaved MBP remains fully in solution.

3) None of the above reaction conditions enhanced solubility without also reducing effective cleavage. Conditions that effectively solubilized the cleaved botulinal C fragment were not identified.

4) The use of 0.1% SDS in the buffer used for Factor Xa cleavage enhanced the solubility of the pMBot protein (all of pMBot protein was soluble). However, the presence of the SDS prevented any cleavage of the fusion protein with Factor Xa.

5) Analysis of pelleted protein from the cleavage reactions indicated that both full length pMBot (i.e., uncleaved) and cleaved botulinal C fragment protein precipitated during incubation.

These results demonstrate that purification of soluble botulinal C fragment protein after cleavage of the pMBot fusion protein is complicated by the insolubility of both the pMBot protein and the cleaved botulinal C fragment protein.

c) Expression Of C. botulinum C Fragment Using Various Expression Vectors

In order to determine if the solubility of the botulinal C fragment was enhanced by expressing the C fragment protein as a native protein, an N-terminal His-tagged protein or as a fusion with glutathione-S-transferase (GST), alternative expression plasmids were constructed. These expression constructs were generated utilizing the methodologies described in Example 22. FIG. 27 provides a schematic representation of the vectors described below.

In FIG. 27, the following abbreviations are used. pP refers to the pET23 vector. pHIS refers to the pETHisa vector. pBlue refers to the pBluescript vector. pM refers to the pMAL-c vector and pG refers to the pGEX3T vector (described in Example 11). The solid black lines represent C. botulinum C fragment gene sequences; the solid black ovals represent the MBP; the hatched ovals represent GST; "HHHHH" (SEQ ID NO: 32) represents the poly-histidine tag. In FIG. 27, when the name for a restriction enzyme appears inside parenthesis, this indicates that the restriction site was destroyed during construction. An asterisk appearing with the name for a restriction enzyme indicates that this restriction site was recreated at a cloning junction.

i) Construction Of pPBot

In order to express the C. botulinum C fragment as a native (i.e., non-fused) protein, the pPBot plasmid (shown schematically in FIG. 27) was constructed as follows. The C fragment sequences present in pAlterBot (Example 22) were removed by digestion of pAlterBot with NcoI and HindIII. The NcoI/HindIII C fragment insert was ligated to pETHisa vector (described in Example 18b) which was digested with NcoI and HindIII. This ligation creates an expression construct in which the NcoI-encoded methionine of the botulinal C fragment is the initiator codon and directs expression of the native botulinal C fragment. The ligation products were used to transform competent BL21(DE3)pLysS cells (Novagen). Recombinant clones were identified by restriction mapping.

ii) Construction Of pHisBot

In order to express the C. botulinum C fragment containing a poly-histidine tag at the amino-terminus of the recombinant protein, the pHisBot plasmid (shown schematically in FIG. 27) was constructed as follows. The NcoI/HindIII botulinal C fragment insert from pAlterbot was ligated into the pETHisa vector which was digested with NheI and HindIII. The NcoI (on the C fragment insert) and NheI (on the pETHisa vector) sites were filled in using the Klenow fragment prior to ligation; these sites were then blunt end ligated (the NdeI site was regenerated at the clone junction as predicted). The ligation products were used to transform competent BL21(DE3)pLysS cells and recombinant clones were identified by restriction mapping.

The resulting pHisBot clone expresses the botulinal C fragment protein with a histidine-tagged N-terminal extension having the following sequence: MetGlyHisHisHisHisHisHisHisHisHisHisSerSerGlyHislleGluGlyArgHisMetAla, (SEQ ID NO:24); the amino acids encoded by the botulinal C fragment gene are underlined and the vector encoded amino acids are presented in plain type. The nucleotide sequence present in the pETHisa vector which encodes the pHisBot fusion protein is listed in SEQ ID NO:25. The amino acid sequence of the pHisBot protein is listed in SEQ ID NO:26.

iii) Construction Of pGBot

The botulinal C fragment protein was expressed as a fusion with the glutathione-S-transferase protein by constructing the pGBot plasmid (shown schematically in FIG. 27). This expression construct was created by cloning the NotI/SalI C fragment insert present in pBlueBot (Example 22) into the pGEX3T vector which was digested with Sinai and XhoI. The NotI site (present on the botulinal fragment) was made blunt prior to ligation using the Klenow fragment. The ligation products were used to transform competent BL21 cells.

Each of the above expression constructs were tested by restriction digestion to confirm the integrity of the constructs.

Large scale (1 liter) cultures of pPBot BL21(DE3)pLysS host!, pHisBot BL21(DE3)pLysS host! and pGBot (BL21 host) were grown in 2X YT medium and induced (using IPTG to 0.8-1.0 mM) for 3 hrs as described in Example 22. Total, soluble and insoluble protein preparations were prepared from 1 ml aliquots of each large scale culture Williams et al. (1994), supra! and analyzed by SDS-PAGE. No obvious induced band was detectable in the pPBot or pHisBot samples by Coomassie staining, while a prominent insoluble band of the anticipated MW was detected in the pGBot sample. Soluble lysates of the pGBot large scale (resuspended in PBS) or pHisBot large scale resuspended in Novagen 1X binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9)! cultures were prepared and used to affinity purify soluble affinity-tagged protein as follows.

The pGBot lysate was affinity purified on a glutathione-agarose resin (Pharmacia) exactly as described in Smith and Corcoran Current Protocols in Molecular Biology, Supplement 28 (1994), pp. 16.7.1-16.7.7!. The pHisBot protein was purified on the His-Bind resin (Novagen) utilizing the His-bind buffer kit (Novagen) exactly as described by manufacturer.

Samples from the purification of both the pGBot and pHisBot proteins (including uninduced, induced, total, soluble, and affinity-purified eluted protein) were resolved on SDS-PAGE gels. Following electrophoresis, proteins were analyzed by Coomassie staining or by Western blot detection utilizing a chicken anti-C. botulinum Type A toxoid antibody (as described in Example 22).

These studies showed that the pGBot protein was almost entirely insoluble under the utilized conditions, while the pHisBot protein was soluble. Affinity purification of the pHisBot protein on this first attempt was inefficient, both in terms of yield (most of the immunoreactive botulinal protein did not bind to the His-bind resin) and purity (the botulinal protein was estimated to comprise approximately 20% of the total eluted protein).

d) Purification Of Soluble C. botulinum C Fragment Protein Substantially Free Of Endotoxin Contamination

The above studies showed that the pHisBot protein was expressed in E. coli as a soluble protein. However, the affinity purification of this protein on the His-bind resin was very inefficient. In order to improve the affinity purification of the soluble pHisBot protein (in terms of both yield and purity), an alternative poly-histidine binding affinity resin (Ni-NTA resin; Qiagen) was utilized. The Ni-NTA resin was reported to have a superior binding affinity (K_(d) =1×10⁻¹³ at pH 8.0; Qiagen user manual) relative to the His-bind resin.

A soluble lysate (in Novagen 1× binding buffer) from an induced 1 liter 2× YT culture was prepared as described above. Briefly, the culture of pHisBot B121(DE3)pLysS host! was grown at 37° C. to an OD₆₀₀ of 0.7 in 1 liter of 2× YT medium containing 100 μg/ml ampicillin, 34 μg/ml chloramphenicol and 0.2% glucose. Protein expression was induced by the addition of IPTG to 1 mM. Three hours after the addition of the IPTG, the cells were cooled for 15 min in a ice water bath and then centrifuged 10 min at 5000 rpm in a JA10 rotor (Beckman) at 4° C. The pellets were resuspended in a total volume of 40 mls Novagen 1× binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9), transferred to two 35 ml Oakridge tubes and frozen at -70° C. for at least 1 hr. The tubes were thawed and the cells were lysed by sonication (4×20 second bursts using a Branson Sonifier 450 with a power setting of 6-7) on ice. The suspension was clarified by centrifugation for 20 rain at 9,000 rpm (10,000 × g) in a JA-17 rotor (Beckman).

The soluble lysate was brought to 0.1% NP40 and then was batch absorbed to 7 ml of a 1:1 slurry of Ni-NTA resin:binding buffer by stirring for 1 hr at 4° C. The slurry was poured into a column having an internal diameter of 1 or 2.5 cm (BioRad). The column was then washed sequentially with 15 mls of Novagen 1× binding buffer containing 0.1% NP40, 15 ml of Novagen 1× binding buffer, 15 ml wash buffer (60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) and 15 ml NaHPO₄ wash buffer (50 mM NaHPO₄, pH 7.0, 0.3 M NaCl, 10% glycerol). The bound protein was eluted by protonation of the resin using elution buffer (50 mM NaHPO₄, pH 4.0, 0.3 M NaCl, 10% glycerol). The eluted protein was stored at 4° C.

Samples of total, soluble and eluted protein were resolved by SDS-PAGE. Protein samples were prepared for electrophoresis as described in Example 22b. Duplicate gels were stained with Coomassie blue to visualize the resolved proteins and C. botulinum type A toxin-reactive protein was detected by Western blot analysis as described in Example 22b. A representative Coomassie stained gel is shown in FIG. 28. In FIG. 28, the following samples were loaded on the 12.5% acrylamide gel. Lanes 1-4 contain respectively total protein, soluble protein, soluble protein present in the flow-through of the Ni-NTA column and affinity-purified pHisBot protein (i.e., protein released from the Ni-NTA resin by protonation). Lane 5 contains high molecular weight protein markers (BioRad).

The purification of pHisBot protein resulted in a yield of 7 mg of affinity purified protein from a 1 liter starting culture of BL21(DE3)pLysS cells harboring the pHisBot plasmid. The yield of purified pHisBot protein represented approximately 0.4% of the total soluble protein in the induced culture. Analysis of the purified pHisBot protein by SDS-PAGE revealed that at least 90-95% of the protein was present as a single band (FIG. 28) of the predicted MW (50 kD). This 50 kD protein band was immunoreactive with anti-C. botulinum type A toxin antibodies. The extinction coefficient of the protein preparation was determined to be 1.4 (using the Pierce BCA assay) or 1.45 (using the Lowry assay) OD₂₈₀ per 1 mg/ml solution.

Samples of pH neutralized eluted pHisBot protein were resolved on a KB 803 HPLC column (Shodex). Although His-tagged proteins are retained by this sizing column (perhaps due to the inherent metal binding ability of the proteins), the relative mobility of the pHisBot protein was consistent with that expected for a non-aggregated protein in solution. Most of the induced pHisBot protein was determined to be soluble under the growth and solubilization conditions utilized above (i.e., greater than 90% of the pHisBot protein was found to be soluble as judged by comparison of the levels of pHisBot protein seen in total and soluble protein samples prepared from BL21(DE3)pLysS cells containing the pHisBot plasmid). SDS-PAGE analysis of samples obtained after centrifugation, extended storage at -20° C., and at least 2 cycles of freezing and thawing detected no protein loss (due to precipitation), indicating that the pHisBot protein is soluble in the elution buffer (i.e., 50 mM NaHPO₄, pH 4.0, 0.3 M NaCl, 10% glycerol).

Determination of endotoxin contamination in the affinity purified pHisBot preparation (after pH neutralization) using the LAL assay (Associates of Cape Cod) detected no significant endotoxin contamination. The assay was performed using the endpoint chromogenic method (without diazo-coupling) according to the manufacturer's instructions. This method can detect concentrations of endotoxin greater than or equal to 0.03 EU/ml (EU refers to endotoxin units). The LAL assay was run using 0.5 ml of a solution comprising 0.5 mg pHisBot protein in 50 mM NaHPO₄, pH 7.0, 0.3 M NaCl, 10% glycerol; 30-60 EU were detected in the 0.5 ml sample. Therefore, the affinity purified pHisBot preparation contains 60-120 EU/mg of protein. FDA Guidelines for the administration of parenteral drugs require that a composition to be administered to a human contain less than 5 EU/kg body weight (The average human body weight is 70 kg; therefore up to 349 EU units can be delivered in a parental dose.). Because very small amount of protein are administered in a vaccine preparation (generally in the range of 10-500 μg of protein), administration of affinity purified pHisBot containing 60-120 EU/mg protein would result in delivery of only a small percentage of the permissible endotoxin load. For example, administration of 10-500 μg of purified pHisBot to a 70 kg human, where the protein preparation contains 60 EU/mg protein, results in the introduction of only 0.6 to 30 EU i.e., 0.2 to 8.6% of the maximum allowable endotoxin burden per parenteral dose (less than 5 EU/kg body weight)!.

The above results demonstrate that endotoxin (LPS) does not copurify with the pHisBot protein using the above purification scheme. Preparations of recombinantly produced pHisBot protein containing lower levels of endotoxin (less than or equal to 2 EU/mg recombinant protein) may be produced by washing the Ni-NTA column with wash buffer until the OD₂₈₀ returns to baseline levels (i.e., until no more UV-absorbing material comes off of the column).

The above results illustrate a method for the production and purification of soluble, botulinal C fragment protein substantially free of endotoxin.

EXAMPLE 25 Optimization Of The Expression And Purification Of pHisBot Protein

The results shown in Example 24d demonstrated that the pHisBot protein is an excellent candidate for use as a vaccine as it could be produced as a soluble protein in E. coli and could be purified free of pyrogen activity. In order to optimize the expression and purification of the pHisBot protein, a variety of growth and purification conditions were tested.

a) Growth Parameters

i) Host Strains

The influence of the host strain utilized upon the production of soluble pHisBot protein was investigated. A large scale purification of pHisBot was performed as described in Example 24d above! using the BL21(DE3) host (Novagen) rather than the BL21(DE3)pLysS host. The deletion of the pLysS plasmid in the BL21(DE3) host yielded higher levels of expression due to de-repression of the plasmid's T7-lac promoter. However, the yield of affinity-purified soluble recombinant protein was very low (approximately 600 μg/liter culture) when purified under conditions identical to those described in Example 24d above. This result was due to the fact that expression in the BL21(DE3) host yielded very high level expression of the pHisBot protein as insoluble inclusion bodies as shown by SDS-PAGE analysis of protein prepared from induced BL21(DE3) cultures (FIG. 29, lanes 1-7, described below). These results demonstrate that the pHisBot protein is not inherently toxic to E. coli cells and can be expressed to high levels using the appropriate promoter/host combination.

FIG. 29 shows a Coomassie blue stained SDS-PAGE gel (12.5% acrylamide) onto which extracts prepared from BL21(DE3) cells containing the pHisBot plasmid were loaded. Each lane was loaded with 2.5 μl protein sample mixed with 2.5 μl of 2× SDS sample buffer. The samples were handled as described in Example 22b. The following samples were applied to the gel. Lanes 1-7 contain protein isolated from the BL21(DE3) host. Lanes 8-14 contain proteins isolated from the BL21(DE3)pLysS host. Total protein was loaded in lanes 1, 2, 4, 6, 8, 10 and 12. Soluble protein was loaded in Lanes 3, 5, 7, 9, 11 and 13. Lane 1 contains protein from uninduced host cells. Lanes 2-13 contain protein from host cells induced for 3 hours. IPTG was added to a final concentration of 0.1 mM (Lanes 6-7), 0.3 mM (Lanes 4-5) or 1.0 mM (Lanes 2, 3, 8-13). The cultures were grown in LB broth (Lanes 8-9), 2× YT broth (Lanes 10-11) or terrific broth (Lanes 1-7, 12-13). The pHisBot protein seen in Lanes 3, 5 and 7 is insoluble protein which spilled over from Lanes 2, 4 and 6, respectively. High molecular weight protein markers (BioRad) were loaded in Lane 14.

A variety of expression conditions were tested to determine if the BL21(DE3) host could be utilized to express soluble pHisBot protein at suitably high levels (i.e., about 10 mg/ml). The conditions altered were temperature (growth at 37° or 30° C.), culture medium (2× YT, LB or Terrific broth) and inducer levels (0.1, 0.3 or 1.0 mM IPTG). All combinations of these variables were tested and the induction levels and solubility was then assessed by SDS-PAGE analysis of total and soluble extracts prepared from 1 ml samples as described in Williams et al., (1994), supra!.

All cultures were grown in 15 ml tubes (Falcon #2057). All culture medium was prewarmed overnight at the appropriate temperature and were supplemented with 100 μg/ml ampicillin and 0.2% glucose. Terrific broth contains 12 g/l bactotryptone, 24 g/l bacto-yeast extract and 100 ml/1 of a solution comprising 0.17M KH₂ PO₄, 0.72M K₂ HPO₄. Cultures were grown in a incubator on a rotating wheel (to ensure aeration) to an OD₆₀₀ of approximately 0.4, and induced by the addition of IPTG. In all cases, high level expression of insoluble pHisBot protein was observed, regardless of temperature, medium or inducer concentration.

The effect of varying the concentration of IPTG upon 2× YT cultures grown at 23° C. was then investigated. IPTG was added to a final concentration of either 1 mM, 0.1 mM, 0.05 mM or 0.01 mM. At this temperature, similar levels of pHis Bot protein was induced in the presence of either 1 or 0.1 mM IPTG; these levels of expression was lower than that observed at higher temperatures. Induced protein levels were reduced at 0.05 mM IPTG and absent at 0.01 mM IPTG (relative to 1.0 and 0.1 mM IPTG inductions at 23° C.). However, no conditions were observed in which the induced pHisBot protein was soluble in this host. Thus, although expression levels are superior in the BL21(DE3) host (as compared to the BL21(DE3)pLysS host), conditions that facilitate the production of soluble protein in this host could not be identified.

These results demonstrate that production of soluble pHisBot protein was achieved using the BL21(DE3)pLysS host in conjunction with the T7-lac promoter.

ii) Effect Of Varying Temperature, Medium And IPTG Concentration And Length Of Induction

The effect growing the host cells in various mediums upon the expression of recombinant botulinal protein from the pHisBot expression construct in the BL21 (DE3)pLysS host! was investigated. BL21 (DE3)pLysS cells containing the pHisBot plasmid were grown in either LB, 2× YT or Terrific broth at 37° C. The cells were induced using 1 mM IPTG for a 3 hr induction period. Expression of pHisBot protein was found to be the highest when the cells were grown in 2× YT broth (see FIG. 29, lanes 8-13).

The cells were then grown at 30° C. in 2× YT broth and the concentration of IPTG was varied from 1.0, 0.3 or 0.1 mM and the length of induction was either 3 or 5 hours. Expression of pHisBot protein was similar at all 3 inducer concentrations utilized and the levels of induced protein were higher after a 5 hr induction as compared to a 3 hr induction.

Using the conditions found to be optimal for the expression of pHisBot protein, a large scale culture was grown in order to provide sufficient material for a large scale purification of the pHisBot protein. Three 1 liter cultures were grown in 2× YT medium containing 100 μg/ml ampicillin, 34 μg/ml chloramphenicol and 0.2% glucose. The cultures were grown at 30° C. and were induced with 1.0 mM IPTG for a 5 hr period. The cultures were harvested and a soluble lysate were prepared as described in Example 18. A large scale purification was performed as described in Example 24d with the exception that except the soluble lysate was batch absorbed for 3 hours rather than for 1 hour. The final yield was 13 mg pHisBot protein/liter culture. The pHisBot protein represented 0.75% of the total soluble protein.

The above results demonstrate growth conditions under which soluble pHisBot protein is produced (i.e., use of the BL21(DE3)pLysS host, 2× YT medium, 30° C., 1.0 mM IPTG for 5 hours).

b) Optimization Of Purification Parameters

For optimization of purification conditions, large scale cultures (3×1 liter) were grown at 30° C. and induced with 1 mM IPTG for 5 hours as described above. The cultures were pooled, distributed to centrifuge bottles, cooled and pelleted as described in Example 24d. The cell pellets were frozen at -70° C. until used. Each cell pellet represented 1/3 of a liter starting culture and individual bottles were utilized for each optimization experiment described below. This standardized the input bacteria used for each experiment, such that the yields of affinity purified pHisBot protein could be compared between different optimization experiments.

i) Binding Specificity (pH Protonation)

A lysate of pHisBot culture was prepared in PBS (pH 8.0) and applied to a 3 ml Ni-NTA column equilibrated in PBS (pH 8.0) using a flow rate of 0.2 ml/min (3-4 column volumes/hr) using an Econo chromatography system (BioRad). The column was washed with PBS (pH 8.0) until the absorbance (OD₂₈₀) of the elute was at baseline levels. The flow rate was then increased to 2 ml/min and the column was equilibrated in PBS (pH 7.0). A pH gradient (pH 7.0 to 4.0 in PBS) was applied in order to elute the bound pHisBot protein from the column. Fractions were collected and aliquots were resolved on SDS-PAGE gels. The PAGE gels were subjected to Western blotting and the pHisBot protein was detected using a chicken anti-C. botulinum Type A toxoid antibody as described in Example 22.

From the Western blot analysis it was determined that the pHisBot protein begins to elute from the Ni-NTA column at pH 6.0. This is consistent with the predicted elution of a His-tagged protein monomer at pH 5.9.

These results demonstrate that the pH at which the pHisBot protein is protonated (released) from Ni-NTA resin in PBS buffer is pH 6.0.

ii) Binding Specificity (Imidazole Competition)

In order to define purification conditions under which the native E. coli proteins could be removed from the Ni-NTA column while leaving the pHisBot protein bound to the column, the following experiment was performed. A lysate of pHisBot culture was prepared in 50 mM NaHPO₄, 0.5 M NaCl, 8 mM imidazole (pH 7.0). This lysate was applied to a 3 ml Ni-NTA column equilibrated in 50 mM NaHPO₄ 0.5M NaCl (pH 7.0) using an Econo chromatography system (BioRad). A flow rate of 0.2 ml/min (3-4 column volumes/hr) was utilized. The column was washed with 50 mM NaHPO₄, 0.5M NaCl (pH 7.0) until the absorbance of the elute returned to baseline. The flow rate was then increased to 2 ml/min.

The column was eluted using an imidazole step gradient in 50 mM NaHPO₄, 0.5M NaCl (pH 7.0)!. Elution steps were 20 mM, 40 mM, 60 mM, 80 mM, 100 mM, 200 mM, 1.0M imidazole, followed by a wash using 0.1 mM EDTA (to strip the nickel from the column and remove any remaining protein). In each step, the wash was continued until the OD₂₈₀ returned to baseline. Fractions were resolved on SDS-PAGE gels, Western blotted, and pHisBot protein detected using a chicken anti-C. botulinum Type A toxoid antibody as described in Example 22. Duplicate gels were stained with Coomassie blue to detect eluted protein in each fraction.

The results of the PAGE analysis showed that most of the non-specifically binding bacterial protein was removed by the 20 mM imidiazole wash, with the remaining bacterial proteins being removed in the 40 and 60 mM imidazole washes. The pHisBot protein began to elute at 100 mM imidazole and was quantitatively eluted in 200 mM imidazole.

These results precisely defined the window of imidazole wash stringency that optimally removes E. coli proteins from the column while specifically retaining the pHisBot protein in this buffer. These results provided conditions under which the pHisBot protein can be purified free of contaminating host proteins.

iii) Purification Buffers And Optimized Purification Protocols

A variety of purification parameters were tested during the development of an optimized protocol for batch purification of soluble pHisBot protein. The results of these analyses are summarized below.

Batch purifications were performed (as described in Example 24d) using several buffers to determine if alternative buffers could be utilized for binding of the pHisBot protein to the Ni-NTA column. It was determined that quantitative binding of pHisBot protein to the Ni-NTA resin was achieved in either Tris-HCl (pH 7.9) or NaHPO₄ (pH 8.0) buffers. Binding of the pHisBot protein in NaHPO₄ buffer was not inhibited using 5 mM, 8 mM or 60 mM imidazole. Quantitative elution of bound pHisBot protein was obtained in buffers containing 50 mM NaHPO₄, 0.3M NaCl (pH 3.5-4.0), with or without 10% glycerol. However, quantitation of soluble affinity purified pHisBot protein before and after a freeze thaw (following several weeks storage of the affinity purified elute at -20° C.) revealed that 94% of the protein was recovered using the glycerol-containing buffer, but only 68% of the protein was recovered when the buffer lacking glycerol was employed. This demonstrates that glycerol enhanced the solubility of the pHisBot protein in this low pH buffer when the eluted protein was stored at freezing temperatures (e.g., -20° C.). Neutralization of pH by addition of NaH₂ PO₄ buffer did not result in obvious protein precipitation.

It was determined that quantitative binding of pHisBot protein using the batch format occurred after 3 hrs (FIG. 30), but not after 1 hr of binding at 4° C. (the resin was stirred during binding). FIG. 30 depicts a Coomaisse blue stained SDS-PAGE gel (7.5% acrylamide) containing samples of proteins isolated during the purification of pHisBot protein from lysate prepared from the BL21(DE3)pLysS host. Each lane was loaded with 5 μl of protein sample mixed with 5 μl of 2× sample buffer and processed as described in Example 22b. Lane 1 comains high molecular weight protein markers (BioRad). Lanes 2 and 3 contain protein eluted from the Ni-NTA resin. Lane 4 comains soluble protein after a 3 hr batch incubation with the Ni-NTA resin. Lanes 5 and 6 contain soluble and total protein, respectively. FIG. 30 demonstrates that the pHisBot protein is completely soluble compare Lanes 5 and 6 which show that a similar amount of the 50 kD pHisBot protein is seen in both; if a substantial amount (greater than 20%) of the pHisBot protein were partially insoluble in the host cell, more pHisBot protein would be seen in lane 6 (total protein) as compared to lane 5 (soluble protein)!. FIG. 30 also demonstrates that the pHisBot protein is completely removed from the lysate after batch absorption with the Ni-NTA resin for 3 hours (compare Lanes 4 and 5).

The reported high affinity interaction of the Ni-NTA resin with His-tagged proteins (K_(d) =1×10⁻¹³ at pH 8.0) suggested that it should be possible to manipulate the resin-protein complexes without significant release of the bound protein. Indeed, it was determined that after the recombinant protein was bound to the Ni-NTA resin, the resin-pHisBot protein complex was highly stable and remained bound following repeated rounds of centrifugation of the resin for 2 min at 1600×g. When this centrifugation step was performed in a 50 ml tube (Falcon), a tight resin pellet formed. This allowed the removal of spent soluble lysate by pouring off the supernatant followed by resuspension of the pellet in wash buffer. Further washes can be performed by centrifugation. The ability to perform additional washes permits the development of protocols for batch absorption of large volumes of lysate with removal of the lysate being performed simply by centrifugation following binding of the recombinant protein to the resin.

A simplified, integrated purification protocol was developed as follows. A soluble lysate was made by resuspending the induced cell pellet in binding buffer 50 mM NaHPO₄, 0.5M NaCl, 60 mM imidazole (pH 8.0)!, sonicating 4×20 sec and centrifuging for 20 min at 10,000 × g. NP-40 was added to 0.1% and Ni-NTA resin (equilibrated in binding buffer) was added. Eight milliliters of a 1:1 slurry (resin:binding buffer) was used per liter of starting culture. The mixture was stirred for 3 hrs at 4° C. The slurry was poured into a column having a 1 cm internal diameter (BioRad), washed with binding buffer containing 0.1% NP40, then binding buffer until baseline was established (these steps may alternatively be performed by centrifugation of the resin, resuspension in binding buffer containing NP40 followed by centrifugation and resuspension in binding buffer). Imidazole was removed by washing the resin with 50 mM NaHPO₄, 0.3M NaCl (pH 7.0). Protein bound to the resin was eluted using the same buffer (50 mM NaHPO₄, 0.3M NaCl) having a reduced pH (pH 3.5-4.0).

A pilot purification was performed following this protocol and yielded 18 mg/liter affinity-purified pHisBot. The pHisBot protein was greater than 90% pure as estimated by Coomassie staining of an SDS-PAGE gel. This represents the highest observed yield of soluble affinity-purified pHisBot protein and this protocol eliminates the need for separate imidazole-containing binding and wash buffers. In addition to providing a simplified and efficient protocol for the affinity purification of recombinant pHisBot protein, the above results provide a variety of purification conditions under which pHisBot protein can be isolated.

EXAMPLE 26 The pHisBot Protein Is An Effective Immunogen

In Example 23 it was demonstrated that neutralizing antibodies are generated in mouse serum after nasal immunization with the pMBot protein. However, the pMBot protein was found to copurify with significant amounts of endotoxin which could not be easily removed. The pHisBot protein, in contrast, could be isolated free of significant endotoxin contamination making pHisBot a superior candidate for vaccine production. To further assess the suitability of pHisBot as a vaccine, the immunogenicity of the pHisBot protein was determined and a comparison of the relative immunogenicity of pMBot and pHisBot proteins in mice was performed as follows.

Two groups of eight BALBc mice were immunized with either pMBot protein or pHisBot protein using Gerbu GMDP adjuvant (CC Biotech). pMBot protein (in PBS containing 10 mM maltose) or pHisBot protein (in 50 mM NaHPO₄, 0.3M NaCl, 10% glycerol, pH 4.0) was mixed with Gerbu adjuvant and used to immunize mice. Each mouse received an IP injection of 100 μl antigen/adjuvant mix (50 μg antigen plus 1 μg adjuvant) on day 0. Mice were boosted as described above with the exception that the route of administration was IM on day 14 and 28. The mice were bled on day 77 and anti-C. botulinum Type A toxoid titers were determined using serum collected from individual mice in each group (as described in Example 23). The results are shown in Table 40.

                                      TABLE 40                                     __________________________________________________________________________     Anti-C. botulinum Type A. Toxiod Serum IgG Titers In Individual Mice           Immunized With pMBot or                                                        pHisBot Protein                                                                Preimmune.sup.1    pMBot.sup.2   pHisBot.sup.2                                 Sample Dilution    Sample Dilution                                                                              Sample Dilution                                       1:25                                                                              1:125      1:25                                                                              1:125      1:25                                                                              1:125                                   Mouse #                                                                             1:50                                                                              0  0   1:6250                                                                             1:50                                                                              0  0   1:6250                                                                             1:50                                                                              0  0   1:620                               __________________________________________________________________________     1                  0.67                                                                              0.19                                                                              0.055                                                                              0.007                                                                              1.57                                                                              0.79                                                                              0.320                                                                              0.093                                                  8  0          4  9                                          2                  1.16                                                                              0.93                                                                              0.254                                                                              0.075                                                                              1.51                                                                              0.82                                                                              0.409                                                                              0.134                                                  1  1          3  9                                          3                  1.36                                                                              0.45                                                                              0.195                                                                              0.041                                                                              1.59                                                                              1.02                                                                              0.453                                                                              0.122                                                  4  8          6  8                                          4                  1.62                                                                              1.18                                                                              0.334                                                                              0.067                                                                              1.55                                                                              0.84                                                                              0.348                                                                              0.090                                                  2  9          2  0                                          5                  1.61                                                                              1.03                                                                              0.289                                                                              0.067                                                                              1.62                                                                              1.58                                                                              0.895                                                                              0.233                                                  2  0          9  0                                          6                  0.91                                                                              0.24                                                                              0.069                                                                              0.013                                                                              1.48                                                                              0.95                                                                              0.477                                                                              0.145                                                  3  2          5  2                                          7                  0.91                                                                              0.23                                                                              0.058                                                                              0.014                                                                              1.52                                                                              0.72                                                                              0.269                                                                              0.069                                                  0  5          4  5                                          8                  0.74                                                                              0.23                                                                              0.058                                                                              0.014                                                                              1.27                                                                              0.42                                                                              0.116                                                                              0.029                                                  7  4          4  7                                          Mean 0.04                                                                              0.02                                                                              0.011                                                                              0.002                                                                              1.13                                                                              0.56                                                                              0.164                                                                              0.037                                                                              1.51                                                                              0.89                                                                              0.411                                                                              0.114                               Titer                                                                               8  1          3  4          8  6                                          __________________________________________________________________________      .sup.1 The preimmune sample represents the average from 2 sets of              duplicate wells containing serum from a individual mouse immunized with        recombinant Staphylococcus enterotoxin B (SEB) antigen. This antigen is        immunologically unrelated to C. botulinum toxin and provides a control         serum.                                                                         .sup.2 Average of duplicate wells.                                       

The results shown above in Table 40 demonstrate that both the pMBot and pHisBot proteins are immunogenic in mice as 100% of the mice (8/8) in each group seroconverted from non-immune to immune status. The results also show that the average titer of anti-C. botulinum Type A toxoid IgG is 2-3 fold higher after immunization with the pHisBot protein relative to immunization with the pMBot protein. This suggests that the pHisBot protein may be a superior immunogen to the pMBot protein.

EXAMPLE 27 Immunization With The Recombinant pHisBot Protein Generates Neutralizing Antibodies

The results shown in Example 26 demonstrated that both the pHisBot and pMBot proteins were capable of inducing high titers of anti-C. botulinum type A toxoid-reactive antibodies in immunized hosts. The ability of the immune sera from mice immunized with either the pHisBot or pMBot proteins to neutralize C. botulinum type A toxoid in vivo was determined using the mouse neutralization assay described in Example 23b.

The two groups of eight BALBc mice immunized with either pMBot protein or pHisBot protein in Example 26 were boosted again one week after the bleeding on day 77. The boost was performed by mixing pMBot protein (in PBS containing 10 mM maltose) or pHisBot protein (in 50 mM NaHPO₄, 0.3M NaCl, 10% glycerol, pH 4.0) with Gerbu adjuvant as described in Example 26. Each mouse received an IP injection of 100 μl antigen/adjuvant mix (50 μg antigen plus 1 μg adjuvant). The mice were bled 6 days after this boost and the serum from mice within a group was pooled. Serum from preimmune mice was also collected (this serum is the same serum described in the footnote to Table 40).

The presence of neutralizing antibodies in the pooled or preimmune serum was detected by challenging mice with 5 LD₅₀ units of type A toxin mixed with 100 μl of pooled serum. The challenge was performed by mixing (per mouse to be injected) 100 μl of serum from each pool with 100 μl of purified type A toxin standard (50 LD₅₀ /ml prepared as described in Example 23b) and 500 μl of gel-phosphate. The mixtures were incubated for 30 min at room temperature with occasional mixing. Each of four mice were injected IP with the mixtures (0.7 ml/mouse). The mice were observed for signs of botulism for 72 hours. Mice receiving toxin mixed with serum from mice immunized with either the pHisBot or pMBot proteins showed no signs of botulism intoxication. In contrast, mice receiving preimmune serum died in less than 24 hours.

These results demonstrate that antibodies capable of neutralizing C. botulinum type A toxin are induced when either of the recombinant C. botulinum C fragment proteins pHisBot or pMBot are used as immunogens.

EXAMPLE 28 Expression and Purification of Recombinant C. difficile Toxin A Proteins Containing the 1870-2680, 1870-2190 and 1960-2680 Interval

Previously others had raised antibodies against C. difficile toxin A by actively immunizing hamsters against a recombinant polypeptide located within the Interval 6 region Lyerly, D. M., et al. (1990) Curr. Microbiol. 21:29!. The structure of the recombinant clone used by Lyerly et al. (1990) Curr. Microbiol. 2I:29! is shown schematically in FIG. 31 as pUC1960-2680.

In FIG. 31, the following abbreviations are used. pP refers to the pET23 vector; pM refers to the pMal-c vector; pGEX refers to the pGEX vector; Trx refers to thioredoxin; pUC refers to the pUC9 vector; A refers to C. difficile toxin A. The numbers refer to the amino acid interval expressed in a given construct. The solid black boxes represent coding regions; the open box at the 3' end of the pUC 1960-2680 construct represents a portion of α-peptide of the lacZ gene which is encoded by vector sequences. The solid ovals represent the MBP. "HHH" represents the poly-histidine tract. The open circles represent thioredoxin. The hatched ovals represent GST.

Using a hamster model of C. difficile associated disease (CDAD) where antibodies are given prophylactically, the Lyerly, et al. antibodies (intra-Interval 6; pUC1960-2680) were only able to partially protect hamsters against C. difficile infection in terms of survival (4 out of 8 animals survived) and furthermore, these antibodies did not prevent diarrhea in any of the animals. Additionally, animals treated with the intra-Interval 6 antibodies Lyerly, et al. (1990), supra! died when treatment was removed. In contrast, Example 16 demonstrated that passive administration of anti-Interval 6 antibodies (anti-pMA1870-2680) prevented diarrhea in 6 out of 7 animals and completely prevented death due to CDAD in the prophylactic treatment model system. Furthermore passive administration of the anti-Interval 6 antibodies provided a long lasting cure (i.e., treatment could be withdrawn without incident).

While the antibodies of Lyerly, et al. were reported to provide some protection against CDAD, the integrity and purity of the recombinant protein expressed from the pUC1960-2680 construct was not reported. The pUC1960-2680 construct potentially expresses the 1960-2680 aa interval of C. difficile toxin A in the pUC9 vector; this interval is nested within the pMA1870-2680 clone (see FIG. 31).

This example involved: (a) construction of pUC1960-2680 and characterization of the expressed protein by Western blot analysis; (b) cloning and expression of the 1960-2680 interval as an affinity tagged protein in pET and pMal vectors and (c) affinity purification and characterization of soluble MBP tagged proteins from clones expressing the 1870-2680, 1870-2190 or 1960-2680 intervals.

a) Construction of pUC1960-2680 and Characterization of Expressed Protein by Western Blot Analysis

The pUC1960-2680 construct contains a 2.1 kb C. difficile toxin A gene fragment encoding 33 of the 38 repeat units; this construct was generated to provide the same recombinant protein utilized by Lyerly et al. (1990) Curr. Microbiol. 21:29! for the generation of anti-C. difficile toxin A antibodies. pUC1960-2680 was constructed as follows. Briefly, the 2.1 kb PstI fragment containing the C. difficile toxin A repeats was removed from pPA1100-2680 (Example 11) and was cloned into pUC9 which had been digested with PstI and dephosphorylated. The dephosphoryation reaction was performed using calf intestinal alkaline phosphatase (CIP) according to the manufacturers instructions (New England Biolabs). Following restriction digestion and CIP-treatment, the reaction products were resolved on an agarose gel, and the appropriate fragments were excised, mixed, and purified utilizing the Prep-a-Gene kit (BioRad). The eluted DNA was ligated, transformed into JM109 competent cells and recombinant clones isolated, and confirmed by restriction digestion using standard techniques of molecular biology. Plasmid DNA was isolated using the QIA-prep spin plasmid kit (Qiagen).

JM109 containing the pUC1960-2680 construct were grown, induced and total and soluble extracts were prepared as described Lyerly et al. (1990) Curr. Microbial. 21:29!. Briefly, a 500 ml culture of Terrific broth was inoculated with pUC1960-2680 (in JM109) and grown at 37° C. to early stationary phase (0.8 OD₆₀₀). IPTG was added to a final concentration of 1 mM and the culture was induced for 2 hrs. A 1 ml aliquot of the culture was withdrawn prior to the addition of IPTG and served as the uninduced sample. Following growth in the presence of the IPTG for 2 hr, another 1 ml aliquot of the culture was withdrawn and served as the induced sample. These 1 ml uninduced and induced samples were treated as follows. The bacteria were pelleted by centrifugation. The cell pellets were resuspended in 100 μl 2× sample buffer (0.125 mM Tris-HCl, pH 6.8, 2 mM EDTA 6% SDS, 20% glycerol, 0.25% bromophenol blue; β-mercaptoethanol was added to 5% before use).

The remaining culture was then processed to prepare total and soluble extracts for analysis. The culture was distributed into 500 ml centrifuge bottles. The bottles were cooled for 15 min in a ice water bath and the cells were pelleted by centrifugation at 5,000 rpm in a Beckman JA10 rotor. The cell pellet was resuspended in 1/10 initial culture volume (i.e., 50 ml) of TBS (0.05M Tris-HCl, 0.15M NaCl, pH 7.5) and distributed to two 35 ml Oakridge tubes. One and one forth milliliters of a 10 mg/ml solution of lysozyme (in TBS) was added to each tube and the mixtures were incubated on ice for 20 min. The two tubes were stored at -70° C. overnight. One of the two tubes from the induced culture was then thawed and sonicated in ice water using four twenty second bursts (Branson Sonifier Model 450 set at level 5-6). The sample was clarified by centrifugation for 20 min at 9000 rpm (Beckman J2-21 rotor), and the soluble lysate filter sterilized through a 0.45 μm filter. Total (before centrifugation) and soluble (after filter sterilization) extracts were prepared for electrophoresis by mixing 4 μ1 extract with 16 μl PBS and 20 μl 2× sample buffer.

The protein samples were resolved by electrophoresis on a 12.5% SDS-PAGE gel and the proteins detected either by Coomassie blue staining (detects all proteins) and Western blot analysis (detects specific proteins) utilizing a goat anti-toxin A specific antibody (TechLabs) as follows. The 12.5% SDS-PAGE gels were loaded with the protein samples. After electrophoresis, the gel was bisected. One half was stained with Coomassie blue and the proteins on the other half were transferred to a solid support for Western blot analysis. Protein transfer was confirmed by Ponceau S staining (as described in Example 12b). The blot was then incubated for 1 hr at 20° C. in PBS containing 0.1% Tween 20 (PBST) and 5% milk (blocking buffer). Then 10 ml of a solution comprising a 1/1000 dilution of an affinity purified goat anti-C. difficile toxin A antibody (Tech Labs) in blocking buffer was added and the blot was incubated for 1 hr at room temperature. The blot was then washed and the presence of the bound anti-C. difficile antibody was detected using a rabbit anti-goat alkaline phosphatase conjugate as secondary antibody as described in Example 3. The resulting Coomassie blue-stained gel and developed Western blot are shown in FIG. 32.

In FIG. 32, the Coomassie blue-stained gel is shown on the left (lanes 1-5 ) and the Western blot is shown on the right (lanes 6-9). The following abbreviations are used: uninduced (U), induced (I), total (T), soluble (S) and broad range molecular weight markers (M; BioRad). The size of the MW markers is indicated by the numbers to the right of lane 5. FIG. 32 shows that no induced bands corresponding to the size expected for the recombinant pUC1960-2680 protein were detectable by Coomassie blue staining. However, Western blot detection of C. difficile toxin A-reactive material revealed a predominant, inducible protein species of the predicted MW for the full length recombinant C. difficile toxin A protein. Although some induced protein is soluble, the majority of the protein is insoluble compare the amount of protein reactive with the antibody present in lanes 8 (total) and 9 (soluble)!. The recombinant protein produced by pUC1960-2680 was also clearly unstable, since breakdown products were detected even in the uninduced (lane 6) or induced (lane 7) culture samples.

b) Cloning and Expression of the 1960-2680 Interval as an Affinity Tagged Protein in pET and pMal Vectors

As shown above, the protein produced by the pUC1960-2680 construct was unstable (i.e., prone to proteolytic degradation) and furthermore, it lacks an affinity tag. The instability of the pUC1960-2680 protein may be due to the presence of the α-peptide of the lacZ gene at the C terminus of the fusion protein; the presence of these sequences on a fusion protein is known to results in the production of an unstable protein. In order to determine whether soluble, stable, affinity purified fusion protein representing the pUC1960-2680 interval could be isolated, the following two constructs were made. The pPA1960-2680 construct contains the 1960-2680 interval of C. difficile toxin A in the pET23c vector (Novagen). The pET23 series of vectors permits the expression of inserted genes as a fusion protein containing a poly-histidine tag or tract at either the C- or N-terminus of the fusion protein; the pPA1960-2680 construct expresses the C. difficile toxin A repeat region as a fusion protein containing a C-terminal poly-histidine tract. The pMA1960-2680 construct contains the 1960-2680 interval of C. difficile toxin A in the pMal-c vector (New England BioLabs) and expresses a fusion protein comprising the MBP at the N-terminus of the fusion protein.

The pPA1960-2680 construct was made as follows. A pUC1960-2680 clone in which the 2.1 kb PstI fragment was in the opposite transcriptional orientation (relative to the direction of transcription through the LacZ sequences on the vector) was isolated using the methods described in section a). The C. difficile toxin A insert was excised by digestion with BamHI and HindIII and the insert was cloned into the pET23c vector (Novagen) digested with BamHI and HindIII as described in section a).

The pMA1960-2680 construct was created by cloning the C. difficile toxin A repeat region of pPA1960-2680 as an NheI-HindIII fragment into the pMal-c vector cleaved with XbaI (XbaI ends are compatible with NheI ends) and HindIII.

Expression of recombinant protein from these two plasmids was assessed in small scale cultures grown at 30° C., utilizing the BL21(DE3) pLysS (pPA1960-2680) or BL21pLysS (pMA1960-2680) hosts. The following conditions were varied: culture broth (2X YT, LB, Terrific broth) and inducer levels (0.1, 0.3 or 1.0 mM IPTG). All combinations of these variables were tested except in Terrific broth, in which a single concentration (1 mM) of IPTG was tested!. The level of recombinant protein expressed upon induction and the solubility of the recombinant protein was assessed by SDS-PAGE analysis of total and soluble extracts (prepared from 1 ml samples as described in Example 25). All cultures were grown in 15 ml tubes (Falcon 2057); all culture medium was prewarmed overnight at the appropriate temperature, and supplemented with 100 μg/ml ampicillin and glucose to 0.2%. Cultures were grown in a incubator on a rotating wheel (to ensure aeration) to an OD₆₀₀ of approximately 0.5-0.7 and induced with the indicated concentration of IPTG.

In all cases, high level expression of insoluble pPA1960-2680 protein was observed, regardless of the broth or inducer concentration employed. The pMA1960-2680 protein was partially soluble under all tested conditions, with maximal levels of soluble protein produced in 2X YT media at the lower inducer concentrations (i.e., 0.1 and 0.3 mM IPTG).

These results demonstrate that the expression of the 1960-2680 interval of C. difficile toxin A in the pPA1960-2680 construct results in the production of insoluble recombinant protein under the conditions tested. The expression of this interval in the pMA1960-2680 construct permitted the expression of some soluble recombinant protein.

c) Affinity Purification and Characterization of Soluble MBP-Tagged Proteins From Constructs Expressing the 1870-2680, 1870-2190 or 1960-2680 Intervals of C. difficile Toxin A

Large scale (1 liter) cultures of the pMal-c vector (i.e., vector lacking an insert), and each of the following recombinant constructs were grown, induced, and soluble protein fractions isolated: pMA1870-2190 (Example 17), pMA1960-2680 (Example 28b) and pMA1870-2680 Example 11; Interval 6; Interval 6 contains amino acid residues 1873 through 2684 (SEQ ID NO:29) of the C. difficile toxin A protein!. The large scale cultures were grown at 32° C. in 2× YT broth and recombinant protein expression was induced by the addition of IPTG to 0.3 mM at OD₆₀₀ of 0.6. The cultures were induced for 4-5 hrs and then the cells were harvested. Soluble protein extracts were prepared and subjected to affinity chromatography to isolate recombinant fusion protein (Example 11d), and analyzed by Coomassie staining and Western analysis as described (Example 11b).

Briefly, soluble extracts were prepared and applied in PBS to an amylose resin (New England Biolabs) column. The column was eluted with PBS containing 10 mM maltose. Protein yields were 40 mg per 1 liter starting volume (i.e., 1 liter cultures) for each recombinant. Protein samples were analyzed by electrophoresis on 7.5% SDS-PAGE gels followed by staining with Coomassie blue and Western blot analysis as described in section a). Protein samples were prepared for electrophoresis by mixing 1 μl total (T) or soluble (S) protein with 4 μl PBS and 5 μl 2× sample buffer, or 5 μl eluted (E) protein and 5 μl 2× sample buffer or 0.5 μl eluted protein, 4.5 μl PBS and 5 μl 2× sample buffer (1/10E). Samples of pMA1870-2680 and pPA1870-2680 (inclusion body preparations described in Example 11) were also resolved on the gel. The samples were heated to 95° C. for 5 min, then cooled and loaded on a 7.5% SDS-PAGE gel. Broad range molecular weight protein markers (BioRad) were also loaded to allow estimation of the MW of identified fusion proteins.

After electrophoresis, protein was detected by staining the gel with Coomassie blue or the proteins were subjected to Western blotting using a goat anti-toxin A antibody (Tech Labs) as described in section a) above. The resulting gel and Western blot are shown in FIG. 33.

In FIG. 33, the Coomaisse blue-stained gel is shown on the left (lanes 1-10) and the Western blot is shown on the right (lanes 1'-10'). Lanes 1-10 and 1'-10' are mirror images of one another and contain the following samples: lanes 1 and 1' contain pMA1870-2190 (T); lanes 2 and 2' contain pMA1870-2190 (E); lanes 3 and 3' contain pMA1960-2680 (T); lanes 4 and 4' contain pMA1960-2680 (S); lanes 5 and 5' contain pMA1960-2680 (E); lanes 6 and 6' contain pMA1960-2680 (1/10E); lanes 7 and 7' contain pMA1870-2680 (E); lanes 8 and 8' contain pMA1870-2680 (1/10E); lanes 9 and 9' contain pPA1870-2680(N/C) (E) pPA1870-2680(N/C) is described in Examples 15 and 29d!; and lanes 10 and 10' contain molecular weight markers.

The results shown in FIG. 33 demonstrate:

1) That the pMA1870-2190 protein was unstable but was at least partially soluble under the growth conditions utilized. The affinity purified pMA1870-2190 preparation does however contain significant concentrations of full length fusion protein (FIG. 33, lane 2).

2) The pMA1960-2680 protein was partially soluble (compare lanes 3' and 4' in FIG. 33) and the integrity of the affinity purified protein (FIG. 33, lanes 5' and 6') was comparable to that of the pMA1870-2680 preparation (FIG. 33, lane 2).

3) The full-length pMA1960-2680, pMA1870-2680 and pPA1870-2680 proteins bind the anti-C. difficile toxin A antibody, while the full-length pMA1870-2190 protein does not (however, smaller breakdown products of the pMA1870-2190 protein do bind to the antibody as shown in FIG. 33, lanes 1' and 2'). This implies that either the epitopes identified by the antibody are present only in the C terminal end of the repeats, or that the antibodies recognize a conformation that cannot form with the N terminal fragment represented in pMA1870-2190. This observation is similar to the lack of reactivity of N-terminal fragments of the C. difficile toxin B gene (pMB1750-1970) with anti-toxin B antibody (Tech Labs) on Western blots seen in Example 19b (FIG. 24).

The results shown above provide a method for the production of affinity purified recombinant C. difficile toxin A protein from the 1870-2190 and 1960-2680 intervals. These results are in contrast to those obtained when using the pUC1960-2680 construct, which was prepared according to the description of Lyerly et al. (1990) Curr. Microbiol. 21:29!. The protein expressed by the pUC1960-2680 construct was mainly insoluble and could not be affinity purified due to the absence of an affinity tag on the recombinant protein.

EXAMPLE 29 Purification of Soluble, Substantially Endotoxin-Free pPA1870-2680(N/C) Protein

For potential utilization as a human vaccine (i.e., to induce active immunity) or as an antigen in a host animal to induce protective antibodies (i.e., antitoxin) for passive immunization of humans, a protein antigen should be 1) easily purified, 2) well characterized and of a high purity, 3) pyrogen poor (when used as a human vaccine), 4) immunogenic and 5) capable of inducing a protective immune response. In the case of the C. difficile toxin A repeat antigen, the protein must be soluble and capable of assuming a conformation which will induce a protective response. As was shown in Example 17, when pPA1870-2680(N/C) protein, which was expressed as insoluble protein inside inclusion bodies, was solubilized with SDS and then used to immunize chickens, no protective anti-toxin A antibodies were produced.

In this example, the recombinant C. difficile toxin A proteins were expressed and evaluated as vaccine candidates using the criteria stated above. This example involved a) evaluation of the utility of affinity purified pMA1870-2680 protein as a vaccine antigen, b)construction, purification and evaluation of the pGA1870-2680 protein, c) development of a protocol for production of soluble pPA1870-2680, d) construction of pPA1870-2680(N) and large scale purification of N, C and N/C his-tagged 1870-2680 protein, e) construction of pPTrxA1870-2680(N) (C)and (N/C), and large scale purification of N, C and N/C his-tagged Trx 1870-2680 proteins, f) large scale affinity purification of pPA1870-2680 and pPB1750-2360 proteins and determination of endotoxin levels and g) construction, large scale affinity purification of pPB1750-2360(N/C) and determination of endotoxin levels.

a) Evaluation of the Utility of Affinity Purified pMA1870-2680 Protein as a Vaccine Antigen

Although the pMA1870-2680 protein (Example 11) was shown to be easily purified, immunogenic and capable of inducing a protective response (Example 17), the ability to use this protein as a vaccine is limited by the poor purity of the affinity purified protein (see FIG. 33, lanes 7' and 8'). It was estimated that only 50% of the affinity purified protein represents full-length fusion protein. The remainder of the proteins in the affinity purified preparation was found to be primarily MBP alone and contaminating E. coli proteins.

In order to assess whether affinity purified pMA1870-2680 protein could be used as a vaccine candidate, the endotoxin content in two independently affinity purified preparations of pMA1870-2680 protein was determined. Pyrogen content in the samples was assayed utilizing the Limulus assay (LAL kit; Associates of Cape Cod) as described in Example 24d. Both samples of affinity purified pMA1870-2680 were found to contain high levels of endotoxin (>50,000 EU/mg purified recombinant protein). As seen in Examples 24a and b, high endotoxin load was determined to be a general feature of affinity purified MBP fusion proteins, or MBP alone. The above results indicate that, using current purification protocols, affinity purified MBP-C. difficile toxin A fusion proteins are not suitable for use as vaccine antigens.

The pMA1870-2680 expression construct was designed to facilitate purification of the toxin A protein from the MBP tag by cleavage of the fusion protein at the engineered Factor Xa cleavage site located between the MBP and toxin A protein domains. The feasibility of obtaining substantially endotoxin-free, soluble recombinant C. difficile toxin A protein by purification of cleaved C. difficile toxin A protein from the MBP-toxin A fusion protein was assessed. Factor Xa (New England Biolabs) was added to the affinity purified pMA1870-2680 protein (0, 0.2, 0.5, 1.0 and 2.5% Factor Xa/pMA1870-2680 protein ratio) in PBS containing 10 mM maltose and the mixtures were incubated for 5.5 and 20 hrs at room temperature. The extent of cleavage was assessed by Coomassie blue staining proteins after electrophoresis on SDS-PAGE gels. The results demonstrated that some cleavage was observed in the 2.5% Factor Xa sample after 20 hrs, but cleavage was only partial. This indicates that cleavage of pMA1870-2680 is not an efficient purification strategy to obtain soluble endotoxin-free C. difficile toxin A repeat protein using the above tested reaction conditions.

b) Construction, Purification and Evaluation of pG1870-2680 Protein

In order to facilitate evaluation of the GST-containing proteins as a means of large scale production of antigens, the C. difficile toxin A repeats were expressed as a fusion with GST. The C. difficile toxin A repeats were isolated by cleavage of pPA1100-2680 (Example 11) with SpeI followed by treatment with the Klenow fragment to fill in the ends; the DNA was then digested with XhoI. The SpeI (Klenow filled)-XhoI fragment was cloned into EcoRI (Klenow filled)-XhoI cleaved pGEX3T vector (Pharmacia) to yield the pGA1870-2680 expression construct.

A large scale (1 liter) 2× YT culture of pGA1870-2680 in BL21 host cells (Novagen)! was grown in 2× YT medium containing 50 μμg/ml ampicillin and induced (using IPTG to 1.0 mM) for 3 hrs at 30° C. as described in Example 28. A soluble lysate of the pGA1870-2680 large scale culture (resuspended in PBS) was prepared, and used to affinity purify soluble affinity tagged protein. The pGA1870-2680 lysate was affinity purified on Glutathione-agarose resin (Pharmacia) as described in Smith and Corcoran, Current Protocols in Molecular Biology, Suppl. 28 (1994) pp. 16.7.1-16.7.7! with the exception that binding of protein to resin was for 1 hr at 4° C. Briefly, following induction of the 1 liter culture for 3 hrs, the cells were collected by centrifugation for 10 min at 5,000 × g at room temperature. The cell pellet was resuspended in 10 ml ice-cold PBS. The cells were then disrupted by sonication as described in Example 24d. Triton X-100 was added to a final concentration of 1% and the sample was well mixed. Insoluble debris was removed by centrifugation of the sample for 5 min at 10,000 × g at 4° C. The supernatant was carefully removed and added to 1 ml of 50% slurry of glutathione-agarose beads (Pharmacia). The mixture was allowed incubate for 1 hr at 4° C. to allow the GST-tagged fusion protein to bind to the resin. The glutathione-agarose beads were then washed by adding 50 ml of ice-cold PBS, mixing and centrifuging for 10 sec at 500 × g at room temperature. The wash step was repeated twice (for a total of 3 washes). The resin was resuspended in 1 ml of ice-cold PBS and transferred to a 1.5 ml microcentrifuge tube. The resin was pelleted by centrifugation for 10 sec at 500 × g at room temperature. The supernatant was removed and the fusion protein was eluted from the washed resin by adding 1 ml of 50 mM Tris-HCl (pH 8.0) and 5 mM reduced glutathione. The tube was mixed gently for 2 min then centrifuged for 10 sec at 500 × g at room temperature. The elution was repeated twice and the supernatants were pooled. The pooled supernatant, containing the eluted fusion protein, was stored in a solution containing 50 mM Tris-HCl (pH 8.0), 5 mM reduced glutathione and 10% glycerol. Endotoxin content of the purified fusion protein was determined using the LAL kit as described in Example 24d.

Samples from the growth, induction and purification steps (uninduced, induced, total, soluble, and affinity purified elution) were resolved on SDS-PAGE gels, and proteins detected by staining with Coomassie blue (as described in Example 28). The fusion protein was found to be partially soluble (i.e., most protein remained in the pellet) and approximately 0.5 mg/liter starting culture of mostly full length protein was affinity purified. The affinity purified preparation contained approximately 5000 EU/mg of affinity purified fusion protein. These results demonstrate that under the above conditions, the pGEX expression system did not facilitate high level production of recombinant C. difficile toxin A fusion protein, and that the recovered protein contained significant endotoxin contamination.

c) Development of a Protocol for Production of Soluble pPA1870-2680

In Example 11 it was shown that, when produced by growth at 37° C., induced pPA1870-2680 protein is almost entirely insoluble. To determine if expression at a lower temperature could enhance solubility, a culture of pPA1870-2680(N/C) was grown at 30° C. and the level of soluble affinity purifiable protein determined. A soluble lysate (in Novagen 1× binding buffer) from an induced 1 liter 2× YT culture was prepared as described below.

Briefly, a culture of pPA1870-2680(N/C) in the BL21(DE3)pLysS host! was grown at 30° C. to an OD₆₀₀ of 0.9 in 1 liter of 2× YT medium containing 100 μg/ml ampicillin, 34 μg/ml chloramphenicol and 0.2% glucose. Protein expression was induced by the addition of IPTG to 1 mM. After a 5 hr induction, the cells were cooled 15 min in a ice water bath and then centrifuged 10 min at 5,000 rpm in a JA10 rotor (Beckman) at 4° C. The pellets were resuspended in a total volume of 40 mls Novagen 1× binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9), transferred to two 35 ml Oakridge tubes and frozen at -70° C. for at least 1 hr. The tubes were thawed and the cells were lysed by sonication (4×20 second bursts) on ice using a Branson Sonifier 450 with a power setting of 6-7. The suspension was clarified by centrifugation for 20 min at 9,000 rpm (10,000 × g) in a JA-17 rotor. The soluble lysate (after addition of NP40 to 0.1%) was batch absorbed to 7 ml of a 1:1 slurry of NiNTA resin (Qiagen): binding buffer 50 mM NaHPO₄, 0.5 M NaCl, 60 mM imidazole (pH 8.0)! by stirring the mixture for 3 hr at 4° C. The slurry was poured into a column having an internal diameter of 1 cm (BioRad), and washed with the following solutions in succession: 15 mls binding buffer containing 0.1%NP40, 15 ml binding buffer, 15 ml wash buffer (40 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). The bound protein was eluted in 200 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9.

Samples of total, soluble, and eluted protein were resolved by SDS-PAGE. Total protein was detected by staining the gel with Coomassie blue. The purification resulted in a yield of 34 mg of affinity purified protein from a 1 liter starting culture (3.2% of the total soluble extract), of which at least 90-95% of the protein was found to migrated as a single band of the predicted MW (90 kd) for the recombinant C. difficile toxin A fusion protein i.e., the pPA1870-2680(N/C) protein!.

These results provide a method, utilizing reduced growth temperature, that facilitates the efficient purification of high levels of soluble recombinant C. difficile toxin A protein utilizing the pPA1870-2680(N/C) expression plasmid.

d) Construction of pPA1870-2680(N) and Large Scale Purification of N, C and N/C His-Tagged 1870-2680 Protein

Expression plasmids that facilitated expression of the 1870-2680 interval of C. difficile toxin A with either a N-terminal his-tag pPA1870-2680 (N)!, a C terminal his-tag pPA1870-2680(C)! or with both N- and C-terminal his-tags pPA1870-2680(N/C)! were evaluated for large scale production and affinity purification of C. difficile toxin A repeat protein.

The features of the pPA1870-2680(C) and pPA1870-2680(N/C) expression vectors was described in Examples 11 and 15. In Example 11, pPA1870-2680(C) was termed pPA1870-2680 and in Example 15, pPA1870-2680(N/C) was termed pPA1870-2680(H). In order to more clearly identify the location of the polyhistidine tract (his-tag) the plasmids are hereinafter referred to with the (C), (N) and (N/C) suffixes. These three expression plasmids were constructed as follows.

pPA1870-2680(C) was constructed by insertion of the C. difficile toxin A repeat containing SpeI-HindIII fragment from pPA1000-2680 (Example 11a) into the pET23b vector (Novagen) cleaved with NheI and HindIII.

The pPA1870-2680(N/C) plasmid was constructed by replacement of the pPA1870-2680(C) promoter region, contained on a BglII-NdeI fragment, with the corresponding BglII-NdeI fragment from the pET16b vector (Novagen).

The pPA1870-2680(N) vector was created by digestion of pPA1870-2680(N/C) at the C-terminal HindIII site followed by treatment with the Klenow enzyme to fill-in the cut ends. The blunted plasmid was then circularized by ligation to create pPA1870-2680(N).

Large scale cultures of pPA1870-2680(N) and pPA1870-2680(C) were grown (using the BL21(DE3)pLysS host), induced and soluble protein was affinity purified and eluted as described in section c) above. In each case 10-20 mg affinity purified protein was recovered and the purified protein was found to be greater than 50% full length fusion protein as estimated by SDS-PAGE analysis. However, the bulk of the pPA1860-2680(C) protein eluted in the 40 mM wash buffer. In an attempt to identify wash conditions which did not result in the elution of significant amounts of the pPA1860-2680(C) protein, the following experiment was performed.

A one liter culture of pPA1870-2680(C) was grown as described above and purified utilizing a phosphate buffer based binding, wash and elution buffers. Cells were resuspended in phosphate binding buffer (50 mM NaPO₄, 0.5M NaCl, 5 mM imidazole, pH 8.0) and sequentially washed in phosphate binding buffer containing either 20, 40, or 200 mM imidazole. Recombinant protein eluted in all three washes (5.5 mg, 12.5 mg and 12 mg total protein, respectively) indicating that the C-terminal his-tagged protein is not retained by the resin at 40 mM imidazole concentrations in either buffer system utilized.

The above results demonstrated that soluble, affinity purified C. difficile toxin A protein was isolated using any of the pPA1870-2680 (N), (C), or (N/C) expression plasmids. p1 e) Construction of pPTrxA1870-2680(N) (C) and (N/C) and Large Scale Purification of N, C and N/C His-Tagged Trx 1870-2680 Proteins

The thioredoxin (Trx) expression system (Invitrogen) has been developed to facilitate soluble expression of normally insoluble or difficult to express proteins. Genes are cloned into the pTrxFus vector and expressed as fusions with the E. coli thioredoxin protein; this linkage often confers the solubility properties of thioredoxin to the fusion protein La Vallie, et al. (1993) Bio/Technology 11:187!. However, the pTrxFus vector has several undesirable properties for an expression vector. All plasmids must be grown in specific strains and growth media since fusion protein expression in this system is inducible by tryptophan. As well, the promoter is not stringently controlled, such that low level expression of fusion protein occurs at reduced temperatures (i.e., 30° C.). Finally, the expression vector does not contain an affinity tag to facilitate high level affinity purification of soluble fusion protein.

To facilitate construction of IPTG-inducible, affinity tagged Trx fusion proteins, the pETHisTrx vector was constructed. The thioredoxin gene of pTrxFus (Invitrogen) was excised as an NdeI-BamHI DNA fragment and was cloned into NdeI-BamHI digested pETHisb vector (Example 18) to created the pETHisTrx vector.

In the pETHisTrx vector, the Trx gene is expressed from the pET16b promoter and contains the pET16b N-terminal leader and his-tag sequence upstream of Trx, and the pET23b polylinker (from the BamHI site) downstream of the Trx gene for construction of C-terminal genetic fusions. Three expression constructs which facilitate expression of a Trx-toxin A 1870-2680 interval fusion, as N, C or N/C terminal his-tags were constructed as follows.

The pPTrxA1870-2680(N/C) construct was constructed by ligation of the NdeI-BamHI (filled) Trx gene (isolated from the pTrxFus vector) and a SpeI (filled)-XhoI fragment containing the C. difficile toxin A 1870-2680 gene isolated from pPA1100-2680 construct (Example 11)! into the NdeI-XhoI cleaved pETHisb vector (the filled BamHI and SpeI sites blunt end ligate together and create an in-frame Trx-C. difficile toxin A fusion).

The above Trx-C. difficile toxin A fusion was excised as an NdeI-HindIII fragment and inserted into NdeI-HindIII cleaved pET23a vector (Novagen) to create pPTrxA1870-2680(C).

The HindIII site of pPTrxA1870-2680(N/C) was cleaved, filled-in by treatment with the Klenow enzyme and religated to create pPTrxA1870-2680(N).

The above constructions were carried using standard techniques of molecular biology as described in Example 29.

Large scale cultures of all three TrxA1870-2680 fusions i.e., pPTrxA1870-2680(C), pPTrxA1870-2680(N) and pPTrxA1870-2680(N/C)! were grown and soluble affinity purified protein was isolated as described in section c) above. In all cases, affinity purified Trx fusion protein yields were similar in terms of solubility, mg/liter culture yields, and purity to the parallel pPA1870-2680 N, C, or N/C constructs described in section d) above.

f) Large Scale Affinity Purification of pPA1870-2680 and pPB1750-2360 Proteins and Determination of Endotoxin Levels

Preparations of affinity purified pPA1870-2680(N/C) (Example 15) and pPB1750-2360 (Example 15b) protein were generated to determine the endotoxin levels in the purified samples. All buffers were filter sterilized and gloves were worn through the preparation of the buffers to reduce buffer-mediated endotoxin contamination of the purified recombinant protein samples. Large scale purifications of pPA1870-2680(N/C) and pPB1750-2360 proteins were performed as follows.

Briefly, cultures of pPA1870-2680(N/C) and pPB1750-2360 in the BL21(DE3)pLysS host! was grown at 30° C. to an OD₆₀₀ of 1.0 in 1 liter of 2× YT medium containing 100 μg/ml ampicillin, 34 μg/ml chloramphenicol and 0.2% glucose. Expression of the recombinant proteins was induced by the addition of IPTG to 0.3 mM. After 5-6 hrs of induction, the cells were for cooled 15 min in a ice water bath and then centrifuged 10 min at 5,000 rpm in a JA10 rotor (Beckman) at 4° C. The cell pellets were frozen at -70° C. overnight and then thawed and resuspended in a total volume of 40 mls binding buffer (5 mM imidazole, 0.5M NaCl, 50 mM NaPO₄, pH 8.0) and transferred to two 35 ml Oakridge tubes. The cells lysed by sonication (8×20 second bursts) on ice using as described in Example 29c. The suspension was clarified by centrifugation for 30 min at 9,000 rpm (10,000 × g) in a JA-17 rotor (Beckman). The supernatant was removed (this constitutes the soluble lysate) and NP40 was added to a final concentration of 1%. The soluble lysate (after addition of NP40 to 0.1%) was batch absorbed to 8 ml of a 1:1 slurry of NiNTA resin (Qiagen): binding buffer by stirring for 3 hr at 4° C. The slurry was centrifuged for 1 min at 500 × g in 50 ml tube (Falcon), resuspended in 5 mls binding buffer containing 0.1% NP40 and poured into a 2.5 cm diameter column (BioRad). The resin was then washed with 20 mls binding buffer containing 0.1% NP40. The column was attached to a UV monitor (ISCO) and was washed with binding buffer until the baseline was established. Following establishment of the baseline absorbance, the column was washed with wash buffer 20 mM (pPB1750-2360) or 40 mM (pPA1870-2680) imidazole, 0.5M NaCl, 50 mM NaPO₄, pH 8.0! until baseline was reestablished. Imidazole was removed by washing the column with 50 mM NaPO₄, 0.3M NaCl, 10% glycerol, pH 7.0, and the bound proteins were eluted in 50 mM NaPO₄, 0.3M NaCl, 10% glycerol, pH 3.5-4.0. Proteins samples from various stages in the purification process were resolved by electrophoresis on an SDS-PAGE gel; the resulting gel is shown in FIG. 34.

FIG. 34 shows a Coomassie blue-stained gel showing the steps of the purification. Protein samples were prepared for electrophoresis by mixing 1 μl total (T) or soluble (S) or soluble protein after binding to NiNTA resin and centrifugation (A) protein with 4 μl PBS and 5 μl 2× SDS-PAGE sample buffer, or 5 μl eluted (E) protein and 5 μl 2× sample buffer. The samples were heated to 95° C. for 5 min, then cooled and loaded onto a 7.5% SDS-PAGE gel. Broad range molecular weight protein markers (M; BioRad) were also loaded, to allow the estimation of the MW of identified fusion proteins. After electrophoresis, protein was detected generally by staining the gel with Coomassie blue. In FIG. 34, lanes 1-4 contain protein from the purification of the pPA1870-2680 protein and lanes 5-8 contain protein from the purification of the pPB1750-2360 protein.

The purification resulted in a yield of approximately 30 mg/liter of affinity purified protein from 1 liter starting cultures (2-2.5% of the total soluble extract) for both proteins, of which at least 90-95% of the protein migrated as a single band of the predicted MW (90 kD) for the recombinant C. difficile toxin A protein. In both cases, most (i.e, greater than 90%) of the induced protein was soluble, and bound the resin quantitatively under the purification conditions utilized.

The endotoxin levels of the purified recombinant proteins was determined using the LAL kit (Example 24d) and was found to be less than 1.0 EU/mg purified protein for pPA1870-2680(N/C), and greater than 250 EU/mg purified protein for pPB1750-2360. The difference in endotoxin levels between these two purified recombinant proteins may reflect the lower stringency wash utilized during the purification of the pPB1750-2360 protein.

g) Construction, Large Scale Affinity Purification of pPB1750-2360(N/C) and Determination of Endotoxin Levels

As shown above, the affinity purified pPB1750-2360 protein contained higher levels of endotoxin than did the purified pPA1870-2680(N/C) protein. The pPB1750-2360 protein contains a poly-histidine tract at the carboxy-terminus while pPA1870-2680(N/C) contains a poly-histidine tract at both the amino- and carboxy-termini. The presence of a poly-histidine tract at both ends of the fusion protein permitted higher stringency wash conditions to be employed during the affinity purification of pPA1870-2680(N/C) as compared to pPB1750-2360 (40 mM imidazole versus 20 mM imidazole, respectively).

In order to produce a fusion protein comprising the 1750-2360 interval of C. difficile toxin B containing poly-histidine tracts at both the amino- and carboxy-termini, pPB 1750-2360(N/C) was constructed as follows. pPB 1750-2360 (Example 15b) was digested with NdeI and XhoI and the 1.5 kb NdeI/XhoI fragment was isolated and inserted into pETHisb vector (Example 18) digested with NdeI and XhoI. Routine procedures were employed for this construction as described in the preceding Examples.

Large scale purification of pPB1750-2360(N/C) was conducted as described above in section f) for the purification of pPB1750-2360 with the exception that the wash buffer contained 40 mM imidazole, 0.5 M NaCl, 50 mM NaPO₄, pH 8.0. Following the wash step, imidazole was removed by washing the column with 50 mM NaPO₄, 0.3M NaCl, 10% glycerol, pH 7.0. The column was then washed with 50 mM NaPO₄, 0.3M NaCl, 10% glycerol, pH 3.0 in an attempt to elute the bound protein. No pPB1750-2360(N/C) was eluted under these conditions.

The large scale purification was then repeated as described above with the exception that following the wash step using the wash buffer containing 40 mM imidazole, 0.5M NaCl, 50 mM NaPO₄, pH 8.0, the bound protein was eluted using a solution containing 200 mM imidazole, 0.5M NaCl, 50 mM NaPO₄, pH 8.0. The imidazole was removed from the eluted protein by dialysis against PBS.

Analysis of the eluted pPB1750-2360(N/C) on SDS-PAGE gels stained with Coomassie blue revealed a single band of the MW expected for the full-length fusion protein.

The endotoxin levels of the purified pPB1750-2360(N/C) protein was determined using the LAL kit (Example 24d). Three separate determinations were conducted and the endotoxin level was found to be 80, 300 or 450 EU/mg of purified recombinant protein. While not limited to any particular mechanism, it is believed that the inconsistent LAL assay results seen with pPB1750-2360(N/C) and the high reading obtained with pPB1750-2360 (see section f) are due to non-specific agglutination of the LAL components by carbohydrate binding moieties present on the C. difficile toxin B sequences present on these proteins. Regardless of whether the actual endotoxin level is 80 or 450 EU/mg purified protein, the affinity purified pPB1750-2360(N/C) preparation represents a substantially endotoxin-free preparation of recombinant protein (Administration of 10 to 500 μg of purified pPB1750-2360(N/C) would result in the introduction of only 4.5 to 225 EU; in a 70 kg human this amount of endotoxin is 1.3 to 64.5% of the maximum permissible dose).

The above results provide a protocol for the affinity purification of substantially endotoxin-free preparations of recombinant C. difficile toxin A and B repeat proteins in high yields.

EXAMPLE 30 Purification of Soluble pPA1870-2680(N/C), pPA1960-2680 and pPA1870-2190 Proteins

In Example 29, methods for the production of soluble, substantially endotoxin-free samples of pPA1870-2680(N), (C) or (N/C) were provided which produced proteins that met the initial criteria set for antigen production, that is the proteins were 1) easily purified 2) well characterized and of a high purity and 3) substantially endotoxin-free. In this example, the ability to produce similarly pure antigen from the pPA1870-2190 or pPA1960-2680 expression constructs was examined. This example involved a) large scale purification of soluble pPA1870-2190 and pPA1960-2680 proteins and b) construction of the pPTrxA1870-2190 vector and large scale purification of soluble pPTrxA1870-2190 protein.

a) Large Scale Purification of Soluble pPA1870-2190 and pPA1960-2680 Proteins

Previous attempts to produce soluble affinity purified protein utilizing the pPA1870-2190 (Example 17a) or pPA1960-2680 (Example 28) vectors were unsuccessful, as assessed by analysis of total and soluble protein produced in small scale cultures. However, the solubility properties of a protein determined utilizing small scale or minicultures may not translate to large scale cultures, due to differences in buffers, sonication conditions, etc. Indeed, the successful expression of soluble, substantially endotoxin-free C. difficile toxin A repeat protein utilizing the pPA1870-2680 N, C or N/C constructs suggested that the conditions utilized to solubilize these proteins might also enhance solubility of the pPA1870-2190 and pPA1960-2680 proteins. This hypothesis was tested as follows.

Large scale cultures of pPA1870-2190 and pPA1960-2680 were grown and soluble protein affinity purified on Ni-NTA resin as described in Example 29c. Both the BL21(DE3) and BL21(DE3)pLysS hosts for pPA1960-2680, and the BL21(DE3)pLysS host for pPA1870-2190 were utilized. The culture of pPA1870-2680(N/C) in the BL21(DE3)pLysS host! was grown at 30° C. to an OD₆₀₀ of 0.9 in 1 liter of 2× YT medium containing 100 μg/ml ampicillin and 0.2% glucose; when the host utilized harbored the pLysS plasmid, 34 μg/ml chloramphenicol was added to the above medium. Protein expression was induced by addition of IPTG to 1 mM. After 5 hrs of induction, the cells were cooled for 15 min in a ice water bath and then centrifuged for 10 min at 5,000 rpm in a JA10 rotor (Beckman) at 4° C. The pellets were resuspended in a total volume of 40 mls Novagen 1× binding buffer (5 mM imidazole, 0.5M NaCl, 20 mM Tris-HCl, pH 7.9), transferred to two 35 ml Oakridge tubes and frozen at -70° C. for at least 1 hr. The tubes were thawed and the cells were lysed by sonication (4×20 second bursts using a Branson Sonifer 450 with a power setting of 6-7) on ice. The suspension was clarified by centrifugation for 20 min at 9,000 rpm (10,000 × g) in a JA-17 rotor (Beckman) at 4° C. The soluble lysate (after addition of NP40 to 0.1%) was batch absorbed to 7 ml of a 1:1 slurry of NiNTA resin (Qiagen): Novagen 1× binding buffer by stirring for 3 hr at 4° C. The slurry was poured into a 1 cm internal diameter column (BioRad), and washed with the following solutions in succession: 15 mls Novagen 1× binding buffer containing 0.1% NP40, 15 ml Novagen 1× binding buffer, 15 ml wash buffer (40 mM imidazole, 0.5M NaCl, 20 mM Tris-HCl, pH 7.9). The bound protein was eluted in 200 mM imidazole, 0.5M NaCl, 20 mM Tris-HCl, pH 7.9.

Samples of total, soluble, and eluted protein (both the 40 mM and 200 mM wash and elution buffers) were resolved by SDS-PAGE. Total protein was detected by Coomassie staining, and C. difficile toxin A-reactive protein (in the case of pPA1960-2680) detected by Western blot detection, utilizing affinity purified goat anti-toxin A antibody as described in Example 28.

The results of these analyses showed that for the pPA1870-2190 protein, only 600 μg protein/liter culture was purified in the 200 mM imidazole elution. The C. difficile toxin A protein was expressed to high levels with this construct, but most of the induced protein was insoluble. As well, the pPA1870-2190 protein represented less than 10% of the total eluted protein. For the pPA1960-2680 construct, total yields of soluble affinity purified protein was either 1 mg B121(DE3)pLysS host! or 200 μg BL21(DE3) host! in the 200 mM elution fraction. Coomassie and Western analysis demonstrated that the pPA1960-2680 protein was expressed to high levels, but that most of the induced protein was insoluble, and that eluted protein preparations contained only approximately 20% C. difficile toxin A-reactive protein.

The above results demonstrate that the conditions utilized to solubilize the pPA1870-2680 protein were not successful in generating solubilized C. difficile toxin A repeat protein expressed by either the pPA1960-2680 or pPA1870-2190 constructs.

b) Construction of the pPTrxA1870-2190 Plasmid and Large Scale Purification of Soluble Protein

To determine if the solubility of recombinant proteins comprising 1870-2680 interval of C. difficile toxin A could be enhanced by utilizing the solubilizing properties of the Trx protein, a fusion construct in which the 1870-2680 interval was expressed as a fusion to thioredoxin (Trx) was constructed.

The pPTrxA1870-2190 construct was made in two steps. First, the 1870-2190 interval was cloned into the pTrxFus vector (Invitrogen). This was accomplished by ligating the KpnI-SalI fragment from pMA1870-2190 which contains the 1870-2190 interval of C. difficile toxin A into the KpnI-SalI cleaved pTrxFus vector. A recombinant clone containing the appropriate DNA fragments was selected and the sequences encoding the Trx-C. difficile toxin A fusion protein were excised utilizing NdeI and SalI, and cloned into the pETHisb vector (Example 18) cleaved with NdeI and XhoI. The resultant construct, pPTrxA1870-2190, comains an N-terminal his-tagged Trx-C. difficile toxin A fusion driven by the pET16b promoter.

Purification of soluble affinity purified Trx-C. difficile toxin A protein from the pPTrxA1870-2190 construct was performed from a large scale culture as described in section a) above. Total, soluble and elution samples were resolved on a 12.5% SDS-PAGE gel and protein was detected by staining with Coomassie blue.

The results of this analysis revealed that the total yield of affinity purified recombinant protein was 2 mg of greater than 50% pure protein in the 200 mM imidazole elution. This yield of 1 mg specific protein (50% of 2 mg total purified protein) represents a ten fold increase over the yield obtained with the pPA1870-2190 construct (10% of 600 μg, or less than 100 μg specific protein) and demonstrates the solubilizing property of the Trx protein. However, the majority of induced protein was insoluble with both constructs (i.e., pPTrxA1870-2190 and pPA1870-2190) and the overall affinity purifiable protein yield with the pPTrxA1870-2190 vector was still less than 20 fold lower that obtained with the pPA 1870-2680 constructs.

EXAMPLE 31 Protection of Hamsters Against C. difficile Disease with Avian Antibodies (IgY) Against Recombinant C. difficile Toxin A and Toxin B

In this example, experiments were performed to determine if orally administered IgY against a recombinant fragment of C. difficile toxin A and/or recombinant C. difficile toxin B can effectively prevent hamsters against C. difficile disease. This example involved a) the immunization of hens with recombinant C. difficile toxin A or B protein, b) purification, detection and quantification of anti-recombinant C. difficile toxin A and toxin B IgY and c) in vivo protection infection study using either anti-recombinant C. difficile toxin A IgY or a mixture of anti-recombinant C. difficile toxin A IgY and anti-recombinant C. difficile toxin B IgY.

a) Immunization of Hens with Recombinant C. difficile Toxin A or B Proteins

Egg-laying Leghorn hens were each immunized with C. difficile toxin A recombinant protein pMA1870-2680 (the 1870-2680 interval of C. difficile toxin A is referred to as Interval A-6) or C. difficile toxin B recombinant pPB1750-2360 (the 1750-2360 interval of C. difficile toxin B is referred to as Interval B-3). Both recombinant proteins were expressed as soluble products and purified as described in Example 28 (pMA1870-2680) and Example 29 (pPB1750-2360). About 1 mg of each recombinant protein was mixed with complete Freund's adjuvant (prepared as described in Example 1) and subcutaneously administered to the hens at multiple sites. The hens were immunized ten times. The first four immunizations were given on Day 1, 14, 21 and 35. The remaining immunizations were then given at 4 week intervals.

b) Purification, Detection and Quantification of Anti-Recombinant C. difficile Toxin A and Toxin B IgY

Eggs were collected about 1 week after the last boost and IgYs were extracted using PEG as described in Example 1. The anti-recombinant C. difficile toxin A and B IgYs were resuspended as a 4× PEG concentrate (i.e., resuspended in 1/4 of the original yolk volume) in 0.1M carbonate buffer, pH 9.5. The total protein concentration of both of the 4× IgY concentrates was 20 mg/ml as judged by absorbance at 280 nm. The relative levels of IgY specific for reactivity with the immunogens were detected by ELISA as follows.

Microtiter plates were coated at 100 μl/well with either 0.05 μg/ml of the recombinant C. difficile toxin A protein, pPA1870-2680 (Example 11) or 1 μg/ml of the recombinant C. difficile toxin B, pPB1750-2360 (Example 18b). The ELISA was performed as described in Example 13c. The results of this analysis revealed that the antibody titers were both greater than 1:125,000. (Antibody titer is defined as the reciprocal of the highest antibody dilution that gives an ELISA signal that is at least 3-fold over pre-immune IgY.) The amount of specific anti-recombinant toxin A and anti-recombinant toxin B IgY was determined by affinity purification as described in Example 15c. The amount of specific anti-recombinant C. difficile toxin A and B antibodies present in the anti-pMA1870-2680 and anti-pPB1750-2360 preparations was determined to be about 160 μg/ml and 200 μg/ml, respectively.

c) In Vivo Protection Infection Study Using Either Anti-Recombinant C. difficile Toxin A IgY or a Mixture of Anti-Recombinant C. difficile Toxin A IgY and Anti-Recombinant C. difficile Toxin B IgY

An in vivo protection study using antibodies raised against pMA1870-2680 (Example 15) and pPB1750-2360 (Example 18b) was performed using the C. difficile-hamster model. This study employed a hamster model which was modified from that used in Example 9, as follows.

Hamsters were predisposed to infection with C. difficile by I.P. administration of 1 mg/100 gm body weight of Clindamycin phosphate (Biomol) in 1 ml of sterile water. The Clindamycin was administered I.P. using a 1 ml tuberculin syringe (Terumo). About 20-24 hours later, the hamsters were each infected orally with 1 ml of saline containing 1×10⁴ C. difficile (ATCC 43596). The C. difficile was grown for about 48 hours on CCFA (C. difficile selective agar) plates (BBL) prior to infection.

Using the above modifications in the hamster model, the time course of infection (in particular, the time of onset of disease) in the hamsters was much more consistent and rapid as compared to the results obtained using the conditions described in Example 9. For the present study, 3 groups of hamsters (Sasco), 8 per group were treated with 2 mls of a 4× concentrate of preimmune or anti-recombinant C. difficile toxin A IgY containing 40 mg of total IgY; the amount of specific anti-recombinant C. difficile toxin A was approximately 400 μg. The third group was treated with 2 mls of an equal mixture of 4× concentration of IgYs to both recombinant C. difficile toxin A and B giving a final specific concentration to each of 2× (the amount of specific anti-recombinant toxin A and B IgY was approximately 200 μg each). The third group, therefore has one-half the amount of specific antibodies to the recombinant C. difficile toxin A compared to the anti-recombinant C. difficile toxin A only treatment.

Hamsters were treated 3 times daily at roughly 4 hour intervals starting 24-hours prior to infection. The hamsters were treated for 5 days. This was about 1 week less than the treatment period employed in Example 9. The outcome of the present prophylactic treatment study is shown in FIG. 35.

In FIG. 35, the percentage cumulative mortality is displayed along the ordinate and the time (in days) is displayed along the abscissa. The treatment period is indicated by the use of the bar between days 0 and 4. The administration of Clindamycin and the inoculation with C. difficile (marked as "Infection" in FIG. 35) is indicated. The solid black squares represent hamsters which received pre-immune IgY; the open squares represent hamsters which received anti-recombinant C. difficile toxin A IgY (anti-Interval A-6) and the solid black diamonds represent hamsters which received a mixture of anti-recombinant C. difficile toxins A and B IgY (anti-Interval A-6/B-3).

The results shown in FIG. 35 demonstrate that under these model conditions, all of the hamsters treated with pre-immune IgY developed diarrhea less than 24-hours post inoculation. One day post inoculation all of the animals were dead in that group. In contrast, using the conditions employed in Example 9, the group treated with pre-immune IgY took several days before the onset of illness was apparent and often not all of the members died from the disease.

As shown in FIG. 35, the hamsters treated with either the anti-recombinant C difficile toxin A IgY (anti-pMA1870-2680) or anti-recombinant C. difficile toxin A (anti-pMA1870-2680) and toxin B (anti-pPB1750-2360) mixture were protected from death; 62% and 88% survived from each group, respectively. Chi-squared analysis of the results in the anti-recombinant C. difficile toxin A and the mixture treated groups was significant compared to the pre-immune treated group, with P values of less than 0.05 and less than 0.005, respectively. Although the results comparing death as an endpoint between two test groups was not significant (P<0.75), diarrhea in the animals receiving the anti-recombinant C. difficile toxin A and B IgY mix was less severe than that seen in the pre-immune control group.

The above results, using a highly aggressive hamster model of CDAD, show that IgYs against a recombinant C. difficile toxin A protein (pMA1870-2680) was protective, but the addition of antibodies against the recombinant C. difficile toxin B (pPB 1750-2360) provided additional protection (i.e., a lessening of the severity of the disease symptoms).

EXAMPLE 32 Treatment of Hamsters with an Established C. difficile Infection with Avian Antibodies (IgY) Against Recombinant C. difficile Toxin A and Toxin B

In order to determine if orally administered IgY against a recombinant C. difficile toxin A protein and/or recombinant C. difficile toxin B can effectively treat hamsters infected with C. difficile, the following experiments were performed. The example involved a) the immunization of hens with recombinant C. difficile toxin A or B proteins b) purification and detection of anti-recombinant C. difficile toxin A and B chicken IgYs c) an in vivo infection study where hamsters were treated with IgYs against either recombinant C. difficile toxin A or recombinant toxin B (Infection study #1). In addition, a mixture of IgY, containing both anti-recombinant toxin A and B was also used to treat hamsters after infection with C. difficile (Infection study #2). The conditions used in infection study #2 were repeated to yield Infection study #3.

a) Immunization of Hens with Recombinant C. difficile Toxin A or B proteins

Egg-laying Leghorn hens were each immunized with the recombinant C. difficile toxin A recombinant protein pMA1870-2680 (Interval A-6) or the C. difficile toxin B recombinant pPB1750-2360 (Interval B-3). Each recombinant comprises the repeat regions of C. difficile toxin A and toxin B. Both recombinant proteins were expressed as soluble proteins utilizing the pMal vector for the toxin A recombinant (Example 15) and pET for the toxin B recombinant (Example 18b).

About 1 mg of each recombinant protein was mixed with 500 μg of Fowl adjuvant (RIBI Immunochemical Research) for the C. difficile toxin A recombinant and or Freund's adjuvant (prepared as described in Example 1) for the C. difficile toxin B recombinant. Each hen was subcutaneously immunized about 7 times at roughly two to four week intervals.

b) Purification and Detection of Anti-Recombinant C. difficile Toxin A and B Chicken IgYs

Eggs were collected about 1 week after the last boost and antibodies were extracted using PEG as described (Example 1). The IgYs were resuspended as a 8× or 4× concentrate (i.e., resuspension at 1/8 or 1/4 yolk volume in 0.1M carbonate buffer, pH 9.5). The relative levels of specific antibodies to the recombinant immunogens was detected by ELISA as described in Example 13c with the following modifications. The 96-well microtiter plate was coated with 0.05 μg/ml of recombinant toxin A protein pPTrxA1870-2680N/C (Example 29e) or 1 μg/ml of toxin B recombinant pPB1750-2360 (Example 18b) at 100 μl/well. The standard ELISA format to detect anti-recombinant C. difficile toxin A or B was performed (Example 13c). Antibody titers by ELISA were both determined to be greater than 1:125,000.

c) In vivo Infection Study

Three infection studies, #1, #2 and #3 were performed using the hamster model described in Example 31.

i) Infection Study #1

In the infection study #1, three separate experimental groups, each consisting of 12 Golden Syrian hamsters (Sasco) weighing approximately 80-90 grams each were used. The animals were housed at 3 per cage and were offered food and water ad libitum throughout the study. The hamster model was conducted as described in Example 31. At the start of the study, each hamster was predisposed to infection by the intra-peritoneal administration of Clindamycin-phosphate (Biomol) at 1 mg/100 gm body weight in 1 ml of water using a 1 ml tuberculin syringe (27 gauge needle). Approximately 24 hours later, each animal was orally challenged, using an 18 gauge feeding needle, with 1 ml of C. difficile, (strain ATCC 43596) with approximately 10³ to 10⁴ organisms in sterile saline. The organisms were grown for 48 hours on CCFA plates (BBL) prior to infection.

Three hours after inoculation (Day 1 ), treatment was initiated for both groups. The groups were each orally treated using an 18 gauge feeding needle to administer 2 mls of a 4× concentrate of either pre-immune IgY or specific immune IgY against either the recombinant C. difficile toxin A (pMA1870-2680; Interval A-6) or toxin B (pPB1750-2360; Interval B-3). On Day 1, the hamsters were treated additionally two more times at 2 hour intervals. On Day 2, through 4 the hamsters were each treated with 2 mls of the respective antibody preparations 3 times daily roughly at 4 hour intervals. Each 2 ml dose contained about 40 mg of IgY of which about 400 μg is specific IgY (determined by affinity purification as described in Example 15c) to the recombinant toxin protein or about 1200 μg of specific anti-C. difficile toxin protein per day. All animals were observed for the onset of diarrhea and death during and after the treatment period. The results are shown in FIG. 36.

In FIG. 36, the percentage cumulative mortality is displayed along the ordinate and the time (in days) is displayed along the abscissa. The treatment period is indicated by the use of the bar between days 1 and 4. The administration of Clindamycin and C. difficile organisms ("Infection") is indicated. The solid black squares represent hamsters which received pre-immune IgY; the open squares represent hamsters which received a 4× preparation of anti-recombinant C. difficile toxin A IgY (anti-Interval A-6) and the solid black diamonds represent hamsters which received a 4× preparation of anti-recombinant C. difficile toxin B IgY (anti-Interval B-3).

The results shown in FIG. 36 demonstrate that half of the hamsters (6/12) treated after infection with antibodies against the C. difficile toxin A recombinant were protected from death from CDAD. The degree of protection in the anti-recombinant C. difficile toxin A group was statistically significant at P<0.025 using Chi-square analysis. Most of the hamsters (10/12) in that group presented with diarrhea. It appeared that at the concentration tested, antibodies against the C. difficile toxin A recombinant was unable to prevent diarrhea in the hamsters. In contrast, all of the pre-immune and anti-recombinant C. difficile toxin B treated hamsters developed diarrhea and died shortly afterward.

The above results demonstrated that IgYs raised against a recombinant C. difficile toxin A protein (pMA1870-2680) can protect the hamsters from death due to CDAD.

ii) Infection Study #2

A second experiment was conducted basically as described above with the exception that a mixture of antibodies to both recombinant C. difficile toxins A and B was tested for the ability to protect hamsters from CDAD. Equal volumes of an 8× concentration of IgYs to both recombinants (pMA1870-2680 and pPB1750-2360) were mixed to give a final concentration to each recombinant equal to 4×. Each dose (2 ml) contained approximately 80 mg/ml protein containing about 400 μg of specific IgY (1% specific anti-C. difficile toxin protein as compared to the total) to each recombinant. The amount of specific anti-recombinant IgY to each toxin recombinant was determined by affinity purification using the respective recombinant protein. The resulting preparation therefore contains the same final concentration of anti-recombinant toxin A used in the previous experiment (section c(i) above) except it contains twice the amount of protein. Because of this difference, an additional test group was set-up and treated with equal volumes of two 8× concentration of anti-recombinant C. difficile toxin A and pre-immune IgY. As a control, a third group of hamsters were treated with an 8× concentrate of only pre-immune IgY. Nine hamsters per group were infected with 1×10⁴ C. difficile organisms (ATCC 43596) and then were treated 4 hours later with 2 mls of either preimmune IgY, anti-recombinant C. difficile toxin A IgY mixed with preimmune IgY or a mixture of anti-recombinant C. difficile toxin A and B IgYs. The animals were treated as described (section c(i) above) at 3 times a day for 4 days. The outcome of this experiment is shown in FIG. 37.

In FIG. 37, the percentage cumulative mortality is displayed along the ordinate and the time (in days) is displayed along the abscissa. The treatment period is indicated by the use of the bar between days 1 and 4. The administration of Clindamycin and C. difficile organisms ("Infection") is indicated. The solid black squares represent hamsters which received an 8× preparation of pre-immune IgY; the open squares represent hamsters which received a mixture of 8× preparations of pre-immune sera and anti-recombinant C. difficile toxin A IgY (anti-Interval A-6) and the solid black diamonds represent hamsters which received a mixture of 8× preparations of anti-recombinant C. difficile toxins A and B IgY (anti-Interval A-6 and B-3).

The results shown in FIG. 37 demonstrate that a mixture of IgYs to both recombinant C. difficile toxin A and B (pMA1870-2680 and pPB1750-2360) completely protected all the hamsters from death from CDAD. Only 1/3 (3 out of 9) of the animals treated with the mixture of anti C. difficile toxin A and B antibodies exhibited diarrhea (one had a very mild case). Hamsters treated with a mixture of anti-recombinant C. difficile toxin A antibodies (anti-Interval A-6) and pre-immune IgY were partially protected with a 56% survival rate. All except one hamster in the anti-Interval A-6/pre-immune IgY group presented with diarrhea. The survival rate in this group, was comparable to the rate seen in infection study #1 (50%) using only anti-recombinant C. difficile toxin A IgY without the addition of pre-immune IgY. This indicated that the addition of preimmune IgY probably had little or no effect (in terms of non-specific protection from proteases in the GI tract) on the effectiveness of the anti-recombinant C. difficile toxin A IgY. As usual, treatment of animals with pre-immune antibodies alone did not protect the hamsters from C. difficile infection and all the hamsters died within 2 days post-infection. The survival rates seen due to administration of the anti-recombinant C. difficile toxin A IgY and the anti-recombinant C. difficile toxins A and B were both statistically significant compared to pre-immune IgY with P values of less than 0.05 and 0.001, respectively. The P-value comparing both recombinant treated groups was less than 0.10.

The survivors in both infection studies #1 and #2 survived lived long-term (i.e., for a period of greater than or equal to 30 days after withdrawal of treatment; animals were euthanized about one month after withdrawal of treatment when the experiment was terminated). Furthermore, no relapse was observed in these animals (relapse is commonly seen in animals, including humans, treated with drugs such as vancomycin or metronidazole to combat C. difficile infection). These results represent the first time antibodies raised against recombinants proteins derived from C. difficile toxins A and B have been shown to be completely effective in animals given a lethal infection with C. difficile.

iii) Infection Study #3

After several more immunizations of the hens with the recombinant C. difficile toxin A alone (pMA1870-2680) and C. difficile toxin A/B recombinants (a mixture of pMA1870-2680 and pPB1750-2360), the in vivo therapeutic study described above (infection study #2) using the mixture of both antibodies was repeated (infection study #3). Three groups of hamsters, each group consisting of 10 members were treated 4 hours post-infection with either pre-immune IgY, anti-recombinant C. difficile toxin A or a mixture of anti-recombinant C. difficile toxin A and B IgYs at the same dosages and times outlined above. The results of this study is shown in FIG. 38.

In FIG. 38, the percentage cumulative mortality is displayed along the ordinate and the time (in days) is displayed along the abscissa. The treatment period is indicated by the use of the bar between days 1 and 4. The administration of Clindamycin and C. difficile organisms ("Infection") is indicated. The solid black squares represent hamsters which received an 8× preparation of pre-immune IgY; the open squares represent hamsters which received a mixture of 8× preparations of pre-immune sera and anti-recombinant C. difficile toxin A IgY (anti-Interval A-6) and the solid black diamonds represent hamsters which received a mixture of 8× preparations of anti-recombinant C. difficile toxins A and B IgY (anti-Interval A-6 and B-3).

As shown in FIG. 38, the hamsters treated with the antibody mixture to both recombinant C. difficile toxins A and B were completely protected from death as shown in the previous experiment (infection study #2) but in addition none of the treated (anti-recombinant toxins A and B) animals presented with diarrhea. While hamsters treated with anti-recombinant C. difficile toxin A were also protected from mortality (only one of ten died) all but one (90%) had diarrhea. All hamsters treated with preimmune IgY developed diarrhea and died within 48-hours of infection.

Prevention against mortality using antibodies to recombinant C. difficile toxin A and both C. difficile toxins A and B was statistically significant (P<0.001), compared to the results obtained using pre-immune antibody. Also, was shown in previous Examples (16 and sections i and ii above), all the treated hamsters survived long-term with no signs of relapse. The prevention of morbidity in the hamsters, which includes presence of diarrhea and weight loss, by treating with anti-recombinant A and B IgY is shown in Table 41.

                  TABLE 41                                                         ______________________________________                                         Interval A-6 and B-3 Antibodies                                                Reduce CDAD Morbidity                                                                       % Animals                                                                      with               % Weight                                       Treatment Group                                                                             Diarrhea   P       Loss.sup.a                                                                            P                                       ______________________________________                                         Pre-Immune   100                NA.sup.b                                       pmA1870-2680 (A-6)                                                                           90        NS.sup.c                                                                               16     <0.001                                  pmA1870-2680 plus                                                              pPB1750-2360 (A-6/B-3)                                                                       0         <0.001  1      NS                                      ______________________________________                                          .sup.a Weight loss of survivors calculated as the difference between the       starting weight and that at termination of treatment.                          .sup.b NA, not applicable.                                                     .sup.c NS, not significant.                                              

As shown in Table 41, the percent weight loss in the survivors treated with the anti-recombinant C. difficile toxin A IgY alone (pMA1870-2680; A-6) compared to the mean weight before infection was about 16%. The hamsters treated with both antibodies to both recombinants (pMA1870-2680 and pPB 1750-2360; A-6/B-3) only lost 1% of their mean starting weight. These results demonstrate that the antibodies raised against the C. difficile toxin A recombinant protected the hamsters from the fatal stage of CDAD but the addition of antibodies to the C. difficile toxin B recombinant was necessary for the prevention of the diarrheal stage associated with CDAD.

EXAMPLE 33 Relapse During In Vivo Treatment of Hamsters Infected with C. difficile Using Vancomycin Therapy

To determine if relapse of C. difficile disease occurs after vancomycin treatment under conditions used in the previous treatment studies, the following experiment was performed.

The conditions employed for the hamster model were identical to the conditions used in Example 32. Three groups of hamsters (Sasco), each group containing 6 members, were treated with 0.2, 1 or 5 mg/kg of vancomycin (Vancomycin HCl, Lilly) in one ml of sterile water. Animals were dosed once per day for 5 days. An additional untreated group was tested as a control. Hamsters were each orally infected with 1×10³ C. difficile organisms (ATCC 43596) and then vancomycin treatment was begun 3 hours post-infection. The outcome of the experiment, twenty days after infection, is shown in FIG. 39.

In FIG. 39, the percentage cumulative mortality is displayed along the ordinate and the time (in days) is displayed along the abscissa. The treatment period is indicated by the use of the bar between days 1 and 5. The administration of Clindamycin and the inoculation with C. difficile organisms (marked as "Infection" in FIG. 39) is indicated. The solid black squares represent hamsters which received no treatment (untreated); the open squares represent hamsters which received 0.2 mg/kg vancomycin; the solid black diamonds represent hamsters which received 1.0 mg/kg vancomycin; and the open diamonds represent hamsters which received 5.0 mg/kg vancomycin.

The results shown in FIG. 39 demonstrate that the hamsters treated with 0.2 mg/kg of vancomycin all died during the course of treatment. Hamsters treated with 1 mg/kg or 5 mg/kg of vancomycin were protected during the period of treatment, but quickly relapsed and most died shortly after the termination of treatment. All of the treated hamsters developed diarrhea and 83% (5/6) of the hamsters treated with 1 mg/kg vancomycin or 100% (6/6) of the hamsters treated with 5 mg/kg vancomycin died 7 days or 9 days after withdrawal of treatment.

This relapse effect using vancomycin as illustrated here or using metronidazole to treat C. difficile infections in the hamster model or in humans is a common occurrence that has been reported frequently. Up to 100% of hamsters and about 25% of humans treated with either of these two drugs relapse. This relapse effect is in marked contrast to the effect shown in the present invention when treating hamsters infected with C. difficile with IgYs raised against either native or recombinant C. difficile toxin A or B. Relapse rarely or never occurs when animals are treated with anti-C. difficile toxin IgY. Thus, the prevention of relapse by the administration of anti-C. difficile toxin IgY represents an important therapeutic advantage over conventional antibiotic treatments.

EXAMPLE 34 Comparison of C. difficile Toxin A Neutralization In Vivo Using IgYs Against Three Different C. difficile Toxin A Repeat-Containing Recombinant Proteins

Three C. difficile toxin A recombinants proteins from the repeat region of C. difficile toxin A were expressed in the pMal-c vector. Antibodies against each were generated and compared for their ability to neutralize C. difficile toxin A in hamsters. The example involved a) immunization of hens, b) purification and detection of anti-recombinant toxin A IgYs and c) C. difficile toxin A neutralization study in hamsters using anti-recombinant toxin A IgYs.

a) Immunization of Hens

Three groups of egg-laying Leghorn hens were immunized with different toxin A recombinants proteins produced in the pMal vector. All were expressed as MBP fusions. They were pMA1870-2190 (Example 17), pMA1960-2680 (Example 28) and pMA1870-2680 (Example 11). The first two recombinants proteins comprise overlapping sub-fragments within the interval contained in the recombinant pMA1870-2680.

Approximately 1 mg of each recombinant protein was given with Freund's adjuvant to each hen. The standard immunization procedure using this adjuvant was performed as described Example 1. The hens were immunized four times at multiple sites using the time intervals described in Example 32a.

b) Purification and Detection of Anti-Recombinant C. difficile Toxin A IgYs

Antibodies were extracted using PEG from eggs collected after at least one week after the last boost. Anti-C. difficile toxin A (CTA) and pre-immune IgYs were also prepared as a controls (as described in Examples and 1, respectively). The IgYs were resuspended in 0.1M carbonate buffer (pH 9.5) at 4× concentration (1/4 the original yolk volume). The levels of specific antibodies from each group was determined by ELISA. Reactivity was determined against the soluble recombinant toxin A protein pPTrx1870-2680. The pPTrx1870-2680 protein does not contain the MBP as do the other 3 immunogens and therefore the ELISA reactivity is specific to only the toxin A recombinant. The standard ELISA protocol was employed (Example 13c). From the ELISA results, all four of the anti-recombinant C. difficile toxin A IgYs were shown to have very similar titers at greater than 1:31,250 compared to the pre-immune IgY.

c) C. difficile Toxin A Neutralization Study in Hamsters Using Anti-Recombinant Toxin A IgYs

The ability of the above recombinant toxin A IgYs (i.e., pMA1870-2190, pMA1960-2680 and pMA1870-2680) to provide protection against C. difficile toxin A was determined in the hamster model. Two groups of hamsters received the anti-pMA1870-2680 IgYs; therefore a total of 6 treatment groups were examined. The assay was performed as described in Example 14.

One ml of IgY was mixed and preincubated for 1 hour with 30 μg of C. difficile toxin A (Tech Labs) then administered orally to 30-40 gm Golden Syrian hamsters (Charles River). Preimmune and CTA IgY (Example 8) served as negative and positive controls, respectively. The animals were observed for 24 hours and the number dead in each group was tallied. The results of the experiment is shown in Table 42.

                  TABLE 42                                                         ______________________________________                                         Generation of Toxin A Neutralizing Antibodies                                  Against Different Toxin A Recombinant Fragments                                Treatment Group  Alive.sup.1                                                                              Dead.sup.1                                          ______________________________________                                         Preimmune        0         5                                                   CTA              5         0                                                   pMA 1870-2190    0         5                                                   pMA 1960-2680    5         0                                                   pMA 1870-2680 a  5         0                                                   pMA 1870-2680 b  3         2                                                   ______________________________________                                          .sup.1 Study outcome after 24 hours.                                     

As shown in Table 42, pre-treatment of C. difficile toxin A with IgY against pMA1870-2680 prevented death in all 5 treated hamsters in the treatment group designated "pMA1870-2680 a" and 3 out of 5 in the treatment group designated "pMA1870-2680 b." Antibodies raised against pMA1870-2680 and the slightly smaller, carboxy-terminal polypeptide, pMA1960-2680, both prevented death in all 5 animals. In contrast, as with preimmune IgY, IgYs raised against the amino-terminal polypeptide pMA1870-2190 had no effect on the prevention of death. As expected, hamsters treated with CTA IgYs were completely protected from the enterotoxic effects of C. difficile toxin A.

EXAMPLE 35 Identification of Adjuvants that Optimally Induce Neutralizing Antibodies Against Native C. difficile Toxin A In Vivo

In order to compare the ability of different adjuvants to invoke neutralizing antibodies against C. difficile toxin A in hens using a recombinant C. difficile toxin A protein as the immunogen, the following experiments were performed. The example involved a) immunization of hens with a recombinant C. difficile toxin A protein using four different adjuvants; b) determination of anti-recombinant C. difficile toxin A IgY titers by ELISA and c) testing the neutralizing ability of the anti-recombinant C. difficile toxin A IgYs against C. difficile toxin A in vivo.

a) Immunization of Hens with a Recombinant C. difficile Toxin A Protein Using Four Different Adjuvants

Eight groups of egg-laying Leghorn hens, each group containing 4 hens, were immunized with either 100 μg or 1 mg of recombinant toxin A protein (pMA1870-2680; Example 11) in combination with four different adjuvants. The four adjuvants tested were: Freund's (GIBCO), Fowl adjuvant LES-STM (here after referred to as the RIBI adjuvant; RIBI Immunochemical Research, Inc.), Gerbu (Biotech) and Quil A (Accurate Chemical). Each adjuvant was tested at both concentrations of antigen. The procedures for preparation and administration for each of the adjuvants were those provided by each manufacturers' protocol. The adjuvant dose in hens was also determined according to manufacturers recommendation if specified.

For immunization with Freund's adjuvant, the standard protocol was used (Example 1). Briefly, 1 volume of antigen were emulsified in 1.2 volumes of either complete Freund's adjuvant for the first immunization or incomplete Freund's for the subsequent boosts. One milliliter of the antigen/Freund's mixture was administered to each hen at four sites. Since Freund's adjuvant contains an oil, the mixing of Freund's adjuvant with the immunogen required vigorous emulsification of the material until solidification using two syringes connected together by a barrel connector. The other three adjuvants (RIBI, Gerbu and Quil A) are aqueous in composition and uniform mixing with the recombinant antigen was far easier as compared to Freund's. Only gentle vortexing was required for mixing the three aqueous adjuvants. The final mixture using these aqueous adjuvants also remained a homogenous liquid allowing easier administration into the hens as compared to using Freund's.

Using the RIBI adjuvant, each hen received 500 μl of the antigen/adjuvant mixture at one site containing 100 μg of adjuvant. The recommended Quil-A dose for guinea pigs and rabbits was 50 μg and 100 μg, respectively. By extrapolating by weight, the hens were each given 75 μg of the Quil A adjuvant at one site in an antigen/adjuvant volume of 500 μl. Using Gerbu material, each hen received 5 μg of adjuvant in 500 μl antigen mixture at one site. The hens were all immunized subcutaneously for 4 times at roughly two-week intervals.

b) Determination of Anti-Recombinant C. difficile Toxin A IgY Titers by ELISA

Anti-recombinant toxin A antibody levels generated using the different adjuvants were compared by ELISA. About 1 week after the last boost, at least 3 eggs from each of the 8 groups along with pre-immune eggs were collected, yolks pooled within the group and IgYs were extracted by PEG as described in Example 1. The purified anti-recombinant toxin A IgYs were then resuspended in PBS at 1× yolk volume. The protein concentration of each of the preparations, determined by absorbance at 280 nm, were all similar at about 4 to 5 mg/ml. The IgY reactivity and titer of each of the individual antibody preparations against pMA1870-2680 were determined by ELISA against a soluble (pPTrxA1870-2680N/C; Example 29) and an insoluble (pPA1870-2190; Example 17a) analog of the C. difficile toxin A 1870-2680 interval. These recombinant C. difficile toxin A analogs were used to coat the microtiter plates for ELISA instead of the recombinant used in the immunization (pMA 1870-2680) as both pPTrxA1870-2680N/C and pPA 1870-2680 were not expressed as fusions with the MBP as was the pMA1870-2680 immunogen. This was done in order to determine antibody reactivity against the toxin portion of the C. difficile toxin A recombinant specifically rather than to the MBP portion of the fusion protein.

The soluble analog pPTrxA1870-2680N/C used to coat the microtiter plate was expressed as a fusion with thioredoxin which aids in solubility and the resulting fusion protein probably exists in a native conformation. The insoluble analog pPA1870-2190, which presumably contains denatured epitopes, was also used to coat microtiter plates. The ELISA reactivity of each IgY to both the soluble and insoluble analogs was tested to determine if there was any preferential reactivity to one or the other analogs when different adjuvants were used for the generation of the IgY.

The standard ELISA protocol described in Example 13c was used with the exception that 20 to 40 fold-less pPTrxA1870-2680N/C protein was used than normal to coat the 96-well microtiter plates (Falcon, Pro-Bind Assay plates) to reduce background. One-hundred μl/well were coated overnight at 4° C. with the soluble pPTrxA1870-2680N/C protein at 0.05 μg/ml or the insoluble protein pPA1870-2680 at 1 μg/ml. The results are shown in FIG. 40.

In FIG. 40, the results of the ELISA reactivity comparing the antibody titer of each of the adjuvant/antigen combinations to either the insoluble (I) or the soluble (S) C. difficile toxin A recombinant is shown. The following abbreviations were utilized: PI (pre-immune); adjuvants were designated as F, R, Q and G for Freund's, RIBI, Quil-A and Gerbu respectively at either 1 mg (1) or 100 μg (100).

In addition, the antibody titer in each group was compared after 3 or 4 immunizations to determine if antibody response has plateaued (indicated by the use of -3 or -4 in FIG. 40). All four adjuvants were able to elicit antibody responses in the hens to varying degrees, but their antibody responses to the soluble or native antigen and insoluble or denatured antigen differed. Freund's adjuvant generated a greater antibody response to the insoluble analog as compared to the soluble one. Almost no reactivity was seen using Freund's adjuvant with 100 μg of antigen to the soluble analog. There was also no difference in response using Freund's to the insoluble analog at either concentration (100 μg or 1 mg) of immunogen while an increase in reactivity to the soluble analog was seen in the higher concentration compared to the lower concentration. In contrast, the antibody reactivity to the soluble analog was generally greater than the insoluble analog using the three other aqueous adjuvants. Antibody reactivities in the ELISA to the soluble analog were about 2-fold higher compared to the insoluble analog. The antibody response improved in the Gerbu, RIBI and Quil-A groups using the increased dose of antigen (1 mg versus 100 μg, and was more pronounced against the soluble analog compared to the insoluble one. The antibody levels to both the insoluble and soluble analog in most of the groups increased after an additional boosting when comparing the 3rd and 4th immunizations.

The results shown in FIG. 40 demonstrate that the immunization of chickens with Freund's adjuvant using a soluble recombinant C. difficile toxin A immunogen elicits antibodies primarily against the insoluble analog. This finding is important if conformational antibodies are required to confer protection in vivo. If conformational antibodies are required, the alternative adjuvants such as Gerbu or RIBI used here would be preferred. The soluble antigen may become denatured during the harsh emulsification process required when using Freund's adjuvant as compared with the other adjuvants. The resulting denatured antigen would then presumably invoke antibodies primarily against an insoluble or non-conformational analog. This effect using Freund's may be overcome by using more antigen for immunization because less of the total is being denatured and a greater amount of native antigen is present. Indeed, there was an increase in soluble analog antibody reactivity at the higher immunogen concentration while there is no difference in insoluble antibody reactivity at both immunogen concentrations.

c) Testing the Neutralizing Ability of the Anti-Recombinant C. difficile Toxin A IgYs Against C. difficile Toxin A In Vivo

The ability of the antibodies raised against the pMA1870-2680 protein generated above using the different adjuvants to neutralize toxin A was compared in vivo. PEG-purified IgYs from eggs from hens immunized with each of the four adjuvants at the 1 mg immunogen concentration were diluted at 0.5× yolk volume in 0.1M carbonate buffer, pH 9.5. This antibody concentration (0.5×) was chosen because it would illustrate the best differentiation in IgY neutralizing capability using the different adjuvants. Pre-immune antibodies also at 0.5× concentration in carbonate were prepared as controls. The antibodies were diluted in carbonate buffer so they could be orally administered with acid less degradation in the stomach.

The IgY protein concentration by absorbance at 280 nm of all of the 0.5× preparations was 2.4-2.5 mg/ml of which 25 to 50 μg/ml was specific antibody against the C. difficile toxin A recombinant protein. An in vivo protection study of hamsters against C difficile toxin A using the five IgY preps was preformed as described in Example 14(c). Five groups, each consisting of 4 male 30-40 gms Golden Syrian hamsters (Charles River). Each hamster was given a mixture of 30 μg of C. difficile toxin A (Tech Labs) in 1 ml of anti-recombinant C. difficile toxin A IgYs or pre-immune IgY. This mixture was first allowed to preincubate for one hour at 37° C. prior to oral administration. The animals were then observed for 24 hours after administration for the presence of diarrhea and death. The results were tabulated and shown in Table 43.

                  TABLE 43                                                         ______________________________________                                         Generation of Toxin A Neutralizing Antibodies                                  Using Different Adjuvants with pMA 1870-2680                                   Treatment Group                                                                              Healthy.sup.a                                                                             Diarrhea.sup.a                                                                            Dead.sup.a                                 ______________________________________                                         Preimmune     0          1          3                                          Freund's      0          0          4                                          Gerbu         4          0          0                                          RIBI          4          0          0                                          Quil A        4          0          0                                          ______________________________________                                          .sup.a Study outcome after 24 hours.                                     

The results shown in Table 43 demonstrate that premixture of C. difficile toxin A with 0.5× anti-recombinant C. difficile toxin A IgYs generated using the Gerbu, RIBI and Quil A adjuvants before administration prevented all overt symptoms and death from the disease in the hamsters. In contrast, all the animals treated with anti-recombinant C. difficile toxin A IgY generated by use of Freund's adjuvant (as a 0.5× antibody preparation) mixed with C. difficile toxin A failed to protect and the hamsters developed diarrhea and died within 24 hours. Three out of four hamsters treated with pre-immune IgY died and the lone survivor had severe diarrhea. These results showed that the anti-recombinant C. difficile toxin A IgYs generated using Gerbu, RIBI and Quil A were able to neutralize the C. difficile toxin A activity in vivo while the Freund's-generated IgY at the same concentration could not. The inability to neutralize C. difficile toxin A by the Freund's-generated anti-recombinant C. difficile toxin A IgY correlates with its low ELISA reactivity against the soluble toxin A analog. In contrast, all of the other adjuvants invoked high antibody levels to the soluble analog and were neutralizing. These results indicated that the neutralizing potential of the antibodies correlated well with their reactivity to the soluble, but not the insoluble analog. The results also indicated that the maintenance of a soluble or conformational C. difficile toxin A immunogen was important in generating neutralizing antibodies. Thus, the choice of an adjuvants such as RIBI or Gerbu was important to retain the conformation of the immunogen which was important in generating anti-C. difficile toxin A antibodies which were protective in vivo.

EXAMPLE 36 In Vivo Neutralization of Toxin A Using Antibodies Against the Recombinant pPA1870-2680 Protein

To determine if the immunization of hens with the C. difficile toxin A recombinant pPA1870-2680(N/C) induced neutralizing antibodies, the following experiment was performed. The example involved a) immunization of hens with the C. difficile toxin A recombinant pPA1870-2680(N/C) using four different adjuvants; b) purification and detection of anti-recombinant IgY; and c) in vivo neutralization study in hamsters using the anti-pPA1870-2680 antibodies incubated with toxin A.

a) Immunization of Hens with the C. difficile Toxin A Recombinant pPA1870-2680 Using Four Different Adjuvants

Egg-laying Leghorn hens were each immunized with the C. difficile toxin A recombinant pPA1870-2680(N/C) (Example 29d). This recombinant protein is expressed in the pET vector and was shown to be capable of isolation in a highly pure form which contained very low levels of endotoxin as compared to the same region expressed in other vectors such as pMal-c (Example 11). These results showed that the pPA1870-2680 recombinant protein would be compatible for use in a vaccine. Accordingly, the ability of pPA1870-2680 to stimulate an antibody response was tested.

Four groups of hens (2 hens/group) were immunized with 100 g of pPA1870-2680(N/C) (purified as described in Example 29d) using 4 different adjuvants. The adjuvants used were: Freund's (GIBCO), Fowl (RIBI) adjuvant (RIBI Immunochemical), Gerbu (Biotech) and Quil A (Accurate Chemical). The amount of each adjuvant used with the recombinant was described in Example 35. The hens were all immunized 4 times at 2 week intervals.

b) Purification and Detection of Anti-Recombinant IgY

The anti-recombinant pPA1870-2680(N/C) levels using the different adjuvants were compared by ELISA. About one week after the last boost, standard PEG preps were prepared from eggs from each group and resuspended at a 4× concentration (all contained about 20 mg/ml IgY) in 0.1M carbonate buffer, pH. 9.5. The standard ELISA protocol (Example 13c) was followed to determine specific antibody reactivity to soluble immunogen pPA1870-2680. The ELISA results are shown in FIG. 41.

In FIG. 41, the absorbance at 410 nm is plotted against the log₁₀ of the dilution of each antibody tested. The solid black squares represent the results of the ELISA using the pre-immune IgY; the open squares, black diamonds, open diamonds and black triangles represent the results of the ELISA using antibodies generated using pPA1870-2680(N/C) (Interval A2) and the following adjuvants: Gerbu (G-A2); Quil A (Q-A2); RIBI (R-A2) and Freund's (F-A2), respectively.

After 4 immunizations, all the hens generated a specific IgY response against the C. difficile toxin A recombinant expressed in the pET vector i.e., pPA1870-2680N/C)!. The response generated by using Freund's, Fowl (RIBI) adjuvant and Quil A were comparable as shown in FIG. 41. A lower antibody response was seen in the Gerbu immunized hens. Interestingly, using the Freund's adjuvant with pPA1870-2680(N/C) gave the highest anti-recombinant activity, whereas in the previous example (Example 35) using the same recombinant region expressed in pMal-c (pMA1870-2680), Freund's adjuvant generated the weakest response. The other adjuvants invoked similar antibody responses comparing both recombinants. These result indicated that the level of antibody response using Freund's adjuvant may depend on what type of antigen is used.

c) In Vivo Neutralization Study in Hamsters Using the Anti-pPA1870-2680(N/C) Antibodies Incubated with C. difficile Toxin A

The ability of antibodies to neutralize C. difficile toxin A in vivo was compared using antibodies raised against pPA1870-2680(N/C) protein generated using the RIBI and Freund's adjuvants. This assay was preformed as described in Example 35c with the exception that the antibodies were diluted to a 2× concentration containing 10 mg/ml of IgY protein. C. difficile toxin A (Tech Labs) was mixed with antibodies generated using Freund's and Fowl (RIBI) adjuvant and orally administered to hamsters. Hamsters treated with pre-immune IgY served as the control. The number of hamsters which were healthy, had diarrhea or were dead 24 hours after administration of the IgYs is shown in Table 44.

                  TABLE 44                                                         ______________________________________                                         Generation of C. difficile Toxin A Neutralizing Antibodies Using               Different Adjuvants with pPA1870-2680                                          Treatment Group                                                                              Healthy    Diarrhea   Dead                                       ______________________________________                                         Preimmune     0          0          4                                          Freund's      4          0          0                                          RIBI          4          0          0                                          ______________________________________                                    

As shown in Table 44, both the Freund's and RIBI adjuvants used in conjunction with pPA1870-2680(N/C) were able to elicit in vivo neutralizing antibodies against C. difficile toxin A as compared to pre-immune IgY. The ability of the antibodies to neutralize C. difficile toxin A shown in this example and in Example 35 appears to correlate well with their ELISA reactivity to a soluble (native) recombinant protein. These results show that the C. difficile toxin A recombinant, pPA1870-2680(N/C), was immunogenic in hens and was capable of generating in vivo neutralizing antibodies; therefore, the pPA1870-2680(N/C) protein is an excellent vaccine candidate.

EXAMPLE 37 Enteric Coating of IgY Raised Against Recombinant C. difficile Toxin A For Oral Delivery

To determine if the avian antibodies (IgYs) raised against recombinant C. difficile toxin A could be enterically-coated and potentially retain in vivo protective abilities, the following experiment was conducted. The example involved a) enteric coating of anti-recombinant C. difficile toxin A antibodies, b) dissolution studies to determine the disintegration kinetics of the enteric-coated IgYs as a function of pH and c) determination of the stability of the antibody reactivity after coating and dissolution by ELISA.

a) Enteric Coating of Anti-Recombinant C. difficile Toxin A Antibodies

Preliminary studies were performed to determine an effective enteric coating process. Enterically-coated avian antibodies should be more resistant to degradation in the stomach compared to antibodies delivered in solution when the route of administration is oral. Intestinal enteric coatings would remain intact at the low pH ranges found in the stomach and therefore the coated IgYs would be able to pass the through stomach undegraded but dissolve at the higher pHs (about 6.0) and release the IgYs in the intestines. An additional property of the enteric films selected for testing is that they are compatible in aqueous solutions instead of organic solvents during the coating process. This property of the enteric film should probably preserve conformation and integrity of the IgY antibody during the coating process. Since the intestines are the site of C. difficile disease, enteric coating of the anti-C. difficile toxin IgY' should concentrate the amount of antibodies available at the site of infection to improve efficacy and reduce the effective dose required as compared to the use of uncoated IgYs.

The anti-C. difficile toxin A antibodies were coated as follows. Sixty grams of lyophilized antibodies against the recombinant C. difficile toxin A protein pMA1870-2680 (Example 11) were prepared. IgYs from eggs collected from hens immunized with the recombinant protein were purified by PEG-precipitation. The IgY pellets after purification were resuspended in 0.1× PBS, pH 7.4 at about 1/4 starting yolk volume (4×) and from 200 to 250 ml volumes were transferred to 600 ml lyophilizing flasks (Labconco). The IgY solutions were flash frozen in the flasks by rotation in an reagent alcohol bath containing dry ice. The frozen antibodies were lyophilized on a Labconco Freeze Dry System/Lyph Lock 4.5 unit operated according to manufacturer's instruction. About 250 mls of the 4× IgY prep yielded about 10 grams of dry material after lyophilization.

The lyophilized IgY was sent to The Coating Place Inc. (Verona, Wis.) for enteric coating. The antibodies were encapsulated using a Wurster coating chamber which is well-suited for coating materials efficiently and uniformly at a small scale in a single operation. Encapsulated IgYs were prepared using two different coating processes. Either a single step direct process or a two-step process using a non-pariel (i.e., a sugar particle of 40-60 mesh size). The lyophilized IgY was either overcoated directly with the film coatings or a two-step method was performed where first the IgY itself was used to overcoat the non-pariel. Then the IgY-coated sugar particle was then overcoated with the enteric film. The use of the sugar particle provides extra bulk necessary to maintain the antibodies in the coating chamber and can aid in a more uniform application of the enteric film.

Two different aqueous enteric films were selected and used with each coating process. The lyophilized IgY was either overcoated with Aquateric (FMC Corp.) or Eudragit® L30D (Rohm Tech Inc). Both of these coatings are water-soluble enteric film coatings that dissolve at pH 6.5 or 5.5, respectively. Both of these enteric films were selected because they fulfill the selection criteria suitable for the needs as described above. Each of the different coating procedures using both enteric films yielded enterically-coated antibodies product. The two-step process using the sugar particle made the entire overcoating procedure in Wurster apparatus technically easier with less loss of material and subsequent greater yields of final product. An enteric coating of approximately a 27-30% by weight was applied to the IgY using the direct method. About 70% of the remaining weight of this enteric-coated material was IgY. About a 32-33% by weight of the enteric coating was achieved in the IgY-overcoated sugar particle. The remaining 67% by weight of the enteric particle was comprised of about 40-50% due to the sugar particle and about 20% the IgY.

b) Dissolution Studies to Determine the Disintegration Kinetics of the Enteric-coated IgYs as a Function of pH

The performance of each of the enterically-coated IgY were tested by determining their dissolution profile. Properly coated IgY particles with intestinal enteric films should remain intact in a gastric solution of pH 1 to 2, but dissolve and release the IgYs into an intestinal solution of pH 7.5. Simulated gastric fluid at about pH 1.2 and simulated intestinal fluid at pH 7.5 were prepared according to USP guidelines except the digestive enzymes were omitted United States Pharmacopeia, Vol. XXII (1990) United States Pharmacopeial Convention, Rockville, Md., pp. 1788-1789!. Ten milligrams of each enteric coated preparation (i.e., Aquateric and Eudragit® coatings) was added per 1 ml of the simulated gastric and intestinal fluids and mixed gently for 1-2 hours at room temperature. An aliquot of the solution was taken at different time points and checked for the presence of protein released in solution. Protein amounts in solution were determined either by absorbance at 280 nm or using a BCA protein assay (Pierce).

The studies demonstrated that the IgY directly coated with both the Aquateric and Eudragit® coatings and the Aquateric-overcoated IgY sugar-particles failed to perform adequately in the dissolution studies. IgYs at both pH 1.2 and 7.5 were released in the solution within minutes after addition of these particles. The dissolution profile for the Aquateric-overcoated IgY sugar particle monitored by absorbance is shown in FIG. 42. The dissolution profile for the Eudragit® overcoated IgY sugar particle is shown in FIG. 43.

In FIG. 42 the absorbance at 280 nm is plotted against time in minutes. The release of the IgY from the Aquateric-overcoated particle in simulated gastric fluid is shown by the solid black squares; release of the IgY from the coated particle in simulated intestinal fluid is shown by the open black squares. Because the Aquateric film itself absorbs UV at a similar wavelength as protein (275-276 nm), UV absorbance at 280 nm cannot be used to accurately quantitate the amount of IgY in solution. Thus, protein at 1 hour (60 min) dissolution was quantitated using the BCA method in order to obtain an accurate determination of the protein concentration.

As shown in FIG. 42, the amount of specific IgY found after dissolution of the Aquateric-overcoated IgY in the two fluids were similar; 4 mg/ml at pH 1.2 and 4.9 mg/ml at pH 7.5. The difference in absorbance shown in FIG. 42 between the gastric and intestinal solutions is due to the presence of more Aquateric film being dissolved in the intestinal solution.

In contrast to the performance of the failed coatings, the Eudragit®-overcoated IgY sugar particle properly opened and released IgY into the solution in the simulated intestinal fluid in a time-dependant manner, while it remained intact in the gastric fluid. The dissolution profile in the gastric and intestinal solutions of the Eudragit®-overcoated IgY sugar particle as a function of time is shown in FIG. 43.

In FIG. 43, the absorbance at 280 nm is plotted against time in minutes. The release of the IgY from the Eudragit®-overcoated particle in simulated gastric fluid is shown by the solid black squares; release of the IgY from the coated particle in simulated intestinal fluid is shown by the open black squares. Since Eudragit® does not absorb UV at the amounts found in the coatings, absorbance values at 280 nm can be directly converted to protein concentration.

As shown in FIG. 43, little or no protein was released in the gastric solution while protein was continually released into the intestinal solution at a linear rate reaching a maximal dissolution after about 2 hours. Ten mg/ml of Eudragit®-overcoated particles yielded from 2 to 2.5 mg/ml of IgY after dissolution. The Eudragit®-overcoated particles in the gastric solution remained intact for long periods of time, even after further incubation at 4° C. for an additional week.

The dissolution profile Eudragit®-overcoated IgY sugar particles was determined under conditions that mimic normal physiological conditions (i.e., simulated travel through the GI tract). The particle was first placed in the gastric solution for 120 minutes followed by an 180 minute incubation in the intestinal solution. Both of these incubations took place with gentle mixing at 37° C. Under these conditions (i.e., incubation in gastric fluid followed by incubation in intestinal fluid), IgY from the Eudragit®-overcoated sugar particle was not released into the gastric solution protein as found in FIG. 42 (i.e., incubation in gastric fluid only), but was only released and detected in the intestinal solution at similar levels found in FIG. 42 (from 2 to 2.5 mg/ml protein released after about 2 hours).

The dissolution studies discussed above demonstrated that the anti-recombinant C. difficile toxin A IgYs were successfully enterically-coated using Eudragit®and a non-pariel.

c) Determination of the Stability of the Antibody Reactivity after Coating and Dissolution by ELISA

The stability of the anti-recombinant C. difficile toxin A IgYs after the overcoating process was determined. This was tested by comparing the ELISA reactivity of the antibodies before coating then after the coating process followed by dissolution at pH 1.2 then pH 7.5. Pre-immune IgY, lyophilized anti-recombinant toxin A IgY starting material and anti-recombinant toxin A IgY obtained from the Eudragit®-overcoated IgY sugar particle after dissolution were first all quantitated for protein and normalized at 2 mg/ml in PBS (pH 7.4). An ELISA was performed detecting antibodies against the recombinant toxin A pPTrxA1870-2680N/C as described in Example 35b. The ELISA results are shown in Table 45.

                  TABLE 45                                                         ______________________________________                                         Comparison of Anti-Recombinant Toxin A                                         Titers by ELISA Before and After Enteric Coating                                                   Pre-Coated Anti-                                                                           Post Coated Anti-                              Dilution                                                                               Preimmune IgY*                                                                             Recombinant A*                                                                             Recombinant A*                                 ______________________________________                                         1:50    0.017       1.4         1.2                                            1:250   0.005       0.59        0.38                                           1:1,250 0.004       0.15        0.10                                           1:6,250 0.005       0.037       0.026                                          1:31,250                                                                               0.007       0.015       0.009                                          1:156,250                                                                              0.009       0.009       0.007                                          ______________________________________                                          *Average A280 readings.                                                  

The results shown in Table 45 demonstrate that the reactivity of the anti-recombinant C. difficile toxin A IgYs before and after Eudragit®-coating to the recombinant C. difficile toxin A protein was very similar. These results indicated that the coating process was not harmful to the IgY and that the IgY remain reactive and functional after dissolution under physiological conditions.

The results shown above demonstrate that enterically-coated IgY that remained stable and active was generated.

EXAMPLE 38 Vaccination of Hamsters Against C. difficile Infection with Recombinant C. difficile Toxin A Proteins

To determine if hamsters vaccinated with C. difficile toxin A recombinant proteins would elicit protective antibodies against C. difficile infection, the following experiment was conducted. Three different C. difficile toxin A recombinants, expressed in the pMal-c or pET vectors, were compared. The example involved a) immunization of hamsters, b) detection of humoral and mucosal anti-recombinant antibody responses by ELISA, and c) challenge study of hamsters with C. difficile.

a) Immunization of Hamsters

Three groups of 90-100 gram female Golden Syrian hamsters (Charles River), each group containing 9 to 11 members, were tested as follows. Hamsters from each group were individually tagged using an ear punch for identification. The animals from each group were housed together and were given food and water ad libitum throughout the course of the experiment. Hamsters were immunized with two different recombinant C. difficile toxin A protein repeats fragments produced the in pMal-c vector and expressed with a maltose binding protein (MBP) fusion and one recombinant C. difficile toxin A protein repeats fragment produced the in pET vector. The animals were immunized subcutaneously with 25 μg of purified protein of either pPA1870-2680N/C (Example 15), pMA1870-2680, a subfragment of pMA1870-2680 called pMA1960-2680 or the MBP (pMal-c) alone as a control. All three recombinant pMal vectors were grown and protein was expressed and purified as described in Example 28c. Recombinant pPA1870-2680N/C was purified as described in Example 29f.

Mixtures comprising 200 μl of antigen and complete Freund's adjuvant (for the first injection) and incomplete Freund's adjuvant (for the subsequent injections) were given subcutaneously behind the neck. The vaccination was administered using a 1 ml 27 gauge tuberculin syringe after the animals were lightly etherized. The animals were vaccinated five times at roughly 2 week intervals.

b) Detection of Humoral and Mucosal Anti-Recombinant Antibody Responses by ELISA

The detection of humoral and mucosal anti-recombinant C. difficile toxin A IgY titers in the hamsters was determined by ELISA. For the humoral response, serum from all members from each group was collected and assayed for anti-recombinant toxin A IgG levels. At least 1 week after the last boost, the hamsters were etherized, bled by cardiac puncture and serum was collected. Ninety-six well microtiter plates (Probind, Falcon) were coated overnight with the soluble C. difficile toxin A recombinant, pPTrxA1870-2680N/C (Example 29e) at 0.05 μg/ml in PBS (pH 7.4) at 100 μl per well. Standard ELISA procedure were followed as described in Example 35b. The secondary antibody used was goat anti-hamster IgG-alkaline phosphatase (Southern Biotech) at a dilution of 1/2000. The average absorbance at 410 nm from duplicate test wells of each serum sample diluted at 1/250 is shown in FIG. 44.

In FIG. 44, the OD₄₁₀ of a 1:250 dilution of serum taken from hamsters immunized with either pMal-c (the pMal-c vector lacking an insert), pMA1870-2680 (Example 28c), pMA1960-2680 (Example 28b) or pPA1870-2680 (Example 15). The numerals shown on the ordinate represent the number assigned to animals within a group.

The results shown in FIG. 44 demonstrate that all the hamsters immunized with the C. difficile toxin A recombinants responded by producing anti-recombinant C. difficile toxin A IgG in the serum. Some variability in the antibody response within the hamsters in a group existed although this difference was not greater than 4-fold. The average antibody response to pMA1960-2680 and pPA1870-2680 was uniformly higher than the response to pMA1870-2680. The hamsters immunized with pMal protein did not produce an anti-serum IgG response to the C. difficile toxin A recombinant protein.

Whether a mucosal IgA response was elicited after immunization was also determined by ELISA. Freshly isolated feces from 4 members of each group were collected, weighed and resuspended by vortexing at 300 μl per 100 mg of stool in PBS, pH 7.4 containing 0.05% thimerosal. The fecal suspension was centrifuged for 5 minutes at 14,000 rpm in a microcentrifuge. Microtiter plates were coated with recombinant antigen as described above. Standard ELISA procedures were used with goat anti-mouse IgA-alkaline phosphatase (Southern Biotech) at 1/1000 as the secondary antibody. This conjugate was used instead of an anti-hamster IgA because the anti-hamster IgA is not commercially available and the anti-mouse antibody has been previously reported to cross-react with hamster IgA. In all samples of fecal extracts, mucosal IgA against recombinant toxin A was not detected by ELISA. These results confirm previous studies Kim and Rolfe (1989) Microbial Ecology in Health and Disease 2:47! in which IgA against toxoid A was not detected in hamsters immunized with a toxoid prepared from C. difficile toxin A.

c) Challenge Study of Hamsters with C. difficile

The vaccinated hamsters (described in section a above) were challenged with C. difficile to determine if the anti-recombinant C. difficile toxin A antibodies were protective against C. difficile disease. Normal hamsters infected with a toxigenic strain of C. difficile develop a fatal disease beginning with diarrhea and eventually die from severe enterocolitis of the cecum (proximal colon) and ileum (as described in Example 9).

The four groups of vaccinated hamsters were first each predisposed with an intra-peritoneal dose of Clindamycin-phosphate (Biomol) in 1 ml of water at 1 mg per 100 gm body weight. About 24 hours later, the hamsters were orally challenged with 1×10⁶ C. difficile in 1 ml of sterile saline using an 18 gauge feeding needle. The animals were lightly anethesized with ether prior to administration. The toxigenic strain of C. difficile, ATCC 43596, was used after 48-hours growth on CCFA plates (BBL). One hamster in the pMA1960-2680 immunized group died accidentally from ether overdose reducing the group number from 9 to 8. The results of the immunization study are shown in Table 46.

                  TABLE 46                                                         ______________________________________                                         Vaccination Against Lethal C. difficile Enterocolitis                          Using Recombinant Toxin A Fragments                                            Vaccination Group    % Protection                                              ______________________________________                                         pMal-c (MBP)         10% (1/10)                                                pMA1960-2680         62% (5/8)                                                 pMA1870-2680         30% (3/10)                                                pPA1870-2680         19% (2/11)                                                ______________________________________                                    

The results shown in Table 46 demonstrate that protection against death occurred in some of the hamsters immunized with each of the recombinant toxin A proteins (i.e., pMA1960-2680 and pMA1870-2680). These results were not statistically significant compared to the fusion control (pMal-c which expresses only the MBP) at a P-value of 0.05 or less using Chi-squared analysis. Ninety percent mortality occurred in the fusion control immunized group (pMal-c). The percent mortality in the pMA1960-2680 immunized group was 38%. The percent mortality in the pMA1870-2680 immunized group was 70% and in the pPA1870-2680 immunized group was 81%. The time to death in recombinant C. difficile toxin A vaccinated group was not delayed compared to the control, occurring up to 3 days after infection. Necropsy of the dead hamsters revealed typical pathology such as severe megacecum.

The specific P-values of the test groups compared to the control group for pMA1960-2680, pMA1870-2680 and pPA1870-2680 groups were less than 0.10, less than 0.75 and less than 0.90, respectively. All of the hamsters except one in the pMA1870-2680 immunized group presented with diarrhea one to two days after infection. There appeared to be no correlation between anti-recombinant C. difficile toxin A antibody titers and the level of protection. For example, hamster number 6 in the pMA1960-2680 immunized group had a lower ELISA titer compared to hamster number 2 (see FIG. 44) yet number 6 survived and number 2 was not protected and died. From these results, hamsters vaccinated with either of the recombinant C. difficile toxin A repeats proteins were not protected against C. difficile-induced diarrhea and from 19 to 62% were protected from the lethal stage of the disease.

The above results correlate with previously published work Lyerly et al. (1990) Curr. Microbiol. 21:29! which showed that hamsters vaccinated with the smaller C. difficile toxin A recombinant fragment (the 1960-2680 interval) expressed in pUC9 could also only partially protect against the lethal stage of disease and none of those hamsters were protected against diarrhea. Lyerly et al. (1990) Curr. Microbiol. 21:29! stated that antibodies to the C. difficile toxin A recombinant protein tested did not prevent the diarrheal stage of the disease and the death in half of the hamsters was due to the varying levels of neutralizing serum antibodies to the toxin A recombinant. From the above results, differences in anti-recombinant C. difficile toxin A titers seen between hamsters in a group may not explain why protection did not occur in all of the animals. The above results indicate that possibly an additional component, possibly a toxin B recombinant protein, is necessary for a more effective vaccine against C. difficile disease.

EXAMPLE 39

Vaccination of Hamsters Against C. difficile Infection with C. difficile Toxin A and Toxin B Recombinant Proteins

Hamsters were immunized with recombinant C. difficile toxin A or recombinant toxin B alone or in combination to test whether this would invoke a humoral response to the recombinant proteins. Furthermore, the ability of the antibodies produced by these vaccinations were tested for the ability to protect the hamsters from infection with C. difficile. Specifically, it was determined if antibodies raised against a recombinant C. difficile toxin B would provide any protection in vivo by itself or above that provided by vaccination with recombinant C. difficile toxin A alone. The example involved a) the immunization of hamsters, b) determination of humoral and mucosal antibody response by ELISA and c) in vivo challenge studies in vaccinated hamsters.

a) Immunization of Hamsters

The recombinant proteins used for vaccination were the C. difficile toxin A recombinant protein pPA1870-2680N/C (Examples 11 and 29) and the C. difficile toxin B recombinant protein pPB1750-2360 (Example 15b). The recombinant proteins were expressed in the pET vector instead of pMal-c vector used in Example 38 because the proteins expressed and isolated using the pET vector were found to be capable of purification at a higher level of purity with lower levels of endotoxin. Production of recombinant proteins in the pET vector is especially amenable for the potential utilization of the recombinant protein as a human vaccine which demands high purity and low levels of potentially harmful endotoxin.

For immunization, 100 μg of pPA1870-2680, 100 μg of pPB1750-2360 or 100 μg of each in combination (200 μg total) were mixed with 2 μg of Gerbu adjuvant (Biotech). The control group were immunized with 100 μg of bovine serum albumin (BSA) with the Gerbu adjuvant. Each group (four total) consisted of 9-10 members of 100 gm female Golden Syrian hamsters (Charles River). Animals were individually tagged to identify members. The hamsters were lightly anesthetized prior to injection sub-cutaneously behind the neck using 1 ml syringe with a 27 gauge needle. The hamsters were immunized 4 times at roughly 2 week intervals.

b) Determination of Humoral and Mucosal Antibody Response by

ELISA

Serum from all individuals from each of the above groups were tested for anti-recombinant protein IgG levels by ELISA. At least one week after the last boost, all of the animals from each group were bled by cardiac puncture and serum was collected. Anti-recombinant C. difficile toxin A and anti-recombinant C. difficile toxin B from the serum samples were determined by ELISA. Ninety-six well microtiter plates (Probind, Falcon) were coated overnight at 4° C. with either pPA1870-2680 protein at 0.05 μg/ml or pPB1750-2360 protein at 1.0 μg/ml in PBS (pH 7.4) at 100 μl per well. Standard ELISA procedures were used exactly as described (Example 13c). The results are shown in FIGS. 45 and 46.

The average absorbance of each serum performed in duplicate and diluted at 1/250 is shown in FIGS. 45 and 46. FIG. 45 shows individual antibody reactivity to the C. difficile toxin A recombinant in the groups immunized with either the C. difficile toxin A recombinant (pPA1870-2680) or a mixture of recombinant C. difficile toxins A and B (pPA1870-2680 and pPB1750-2360). FIG. 46 shows antibody reactivity to recombinant C. difficile toxin B in the groups immunized with either the C. difficile toxin B recombinant (pPB1750-2360) or a mixture of recombinant C. difficile toxins A and B (pPA1870-2680 and pPB1750-2360).

The results shown in FIGS. 45 and 46 demonstrate that in all cases each animal responded and produced a specific IgG antibody response to the immunogen. As expected, the hamsters immunized with BSA (negative control group) did not invoke any antibody response to the recombinant antigens. The anti-recombinant C. difficile toxin A or B response within members of the same group were similar.

The determination of a mucosal anti-recombinant C. difficile toxin A or recombinant C. difficile toxin B IgA response was elicited after immunization was also determined by ELISA. Freshly isolated feces from 4 members of each group were collected, weighed and processed as described in Example 21. Plates were coated with recombinant C. difficile toxin A or recombinant C. difficile toxin B antigen as described above for determination of serum IgG levels. Standard ELISA procedures (Example 13c) were used in conjunction with goat anti-mouse IgA-alkaline phosphatase (Southern Biotech, Birmingham, Ala.). In all samples of fecal extracts, IgA against recombinant toxin A or B was not detected. Again this result using different recombinants confirms that found in Example 38 and with previous studies Kim and Rolfe (1989), supra!.

c) In Vivo Challenge Studies in Vaccinated Hamsters

The vaccinated hamsters described above in section a) above were challenged with C. difficile to determine whether the serum antibody response to either recombinant C. difficile toxin A or B alone or in combination was protective against CDAD. The four groups of vaccinated hamsters were first each predisposed to CDAD with an intra-peritoneal dose of Clindamycin-phosphate (Biomol) in 1 ml of water at 1 mg per 100 gm weight. About 24 hours later, the hamsters were orally challenged with 1×10⁶ C. difficile organisms in 1 ml of sterile saline using an 18 gauge feeding needle. The animals were lightly anethesized with ether prior to administration. The toxigenic strain ATCC 43596 was used after 48-hours growth on CCFA plates (BBL). The results of the immunization study is shown in Table 47.

                  TABLE 47                                                         ______________________________________                                         Vaccination Against Lethal C. difficile Enterocolitis                          Using Recombinant Toxin A and Toxin B Polypeptides                             Vaccination Group.sup.a                                                                             % Protection                                              ______________________________________                                         BSA                   0% (0/10)                                                pPA1870-2680N/C       20% (2/10)                                               pPB1750-2360          0% (0/10)                                                pPA1870-2680N/C & pPB1750-2360                                                                      100% (9/9)                                                ______________________________________                                          .sup.a Vaccinated with 100 μg recombinant protein per hamster               subcutaneously 4 times at 2 week intervals.                              

As shown in Table 47, one to three days after challenge with C. difficile, all of the hamsters immunized with either pPA1870-2680 or pB1750-2360 and the BSA control group developed diarrhea. All the hamsters in those three groups except two members immunized with pPA1870-2680, died from several hours to 48 hours after the detected onset of diarrhea. Necropsy revealed severe enterocolitis in the animals with inflamed and enlarged cecums characteristic of C. difficile disease. In contrast, hamsters immunized with the vaccine comprising the combination of pPA1870-2680 or pB1750-2360 proteins showed no signs of illness such as diarrhea and remained healthy for the entire 14-day post-infection observation period. In fact, these animals have remained healthy for a period of at least 5 months post-infection; these results demonstrate that vaccination with the combination of pPA1870-2680 or pB1750-2360 proteins confers complete and long term protection on hamsters inoculated with C. difficile.

The protective effect seen with the combination vaccine was not due to differences in antibody titer in this group compared to the antibody titers in the hamsters vaccinated with only recombinant C. difficile toxin A or C. difficile toxin B. Protection of the hamsters immunized with the C. difficile toxin A/B combination (i.e., pPA1870-2680 and pB1750-2360) was statistically significant compared to the control; the P value was determined to be less than 0.001.

The above results demonstrate that recombinant C. difficile toxin A and toxin B proteins are both key components for an effective vaccine against C. difficile and that ellictation of antibodies against recombinant C. difficile toxins A or B alone was not sufficient to confer complete protection. Antibodies generated against a recombinant C. difficile toxin B in addition to recombinant C. difficile toxin A both confer protection and they both act synergistically to neutralize C. difficile-associated diarrhea and death. While the invention is not limited by any particular mechanism, the protection from the anti-C. difficile toxin serum antibodies may result from the leakage of the C. difficile toxin A and B neutralizing antibodies into tissues or the intestinal lumen during the inflammation that accompanies the early stages of C. difficile enterocolitis.

The results shown above (vaccination of hamsters with recombinant C. difficile toxins A and B) and in Example 32(c)(iii) (administration of antitoxin comprising a mixture of antibodies raised against both C. difficile toxins A and B) strongly support one another. Together they demonstrate that full protection from CDAD (i.e., protection from both morbidity and mortality) requires the use of recombinant proteins derived from both C. difficile toxins A and B for either active or passive immunization.

EXAMPLE 40 In Vivo Protection Against C. difficile Infection by the Parenteral Administration of Antibodies Against Recombinant C. difficile Toxin A and B Proteins

The results shown in Example 39 demonstrated that vaccination of hamsters with recombinant C. difficile toxin A and B proteins generated neutralizing serum antibodies in the recipient animals which conferred complete protection (i.e., protection from both morbidity and mortality) from the deleterious effects of infection with C. difficile. Example 38 demonstrated that vaccination of hamsters with recombinant C. difficile toxin A proteins produced neutralizing serum anti-toxin A antibodies (IgG) but undetectable levels of mucosal (IgA) anti-toxin A antibodies. Thus, the production of serum anti-toxin A and B antibodies is sufficient to confer protection from CDAD. In order to determine whether parenteral delivery of anti-recombinant toxin A and B IgYs is an effective way to treat C. difficile infection, the following experiment is conducted.

Six groups of 80-100 gram female Golden Syrian hamsters (Charles River), each group containing 9-10 members, are infected with C. difficile as described in Example 32c). The animals are housed three per cage and are offered food and water ad libitum throughout the study. At the start of the study, each hamster is predisposed to infection by the intra-peritoneal administration of Clindamycin-phosphate (Biomol) at 1 mg/100 gram body weight in 1 ml of water using a 1 ml tuberculin syringe (27 gauge needle). Approximately 24 hours later, each animal is orally challenged, using an 18 gauge feeding needle, with 1 ml of C. difficile, (strain ATCC 43596) with approximately 10³ to 10⁴ organisms in sterile saline. The organisms are grown for 48 hours on CCFA plates (BBL) prior to infection.

Three hours after infection (Day 1), treatment is initiated as follows. Each hamster receives 2 mls of a solution comprising either pre-immune IgY (as an 8× PEG preparation) or a mixture of anti-recombinant toxins A and B (e.g., antitoxin raised against pMA1870-2680 and pPB1750-2360). The 8× PEG preparations are prepared and mixed as described in 32(c)(ii) with the exception that the IgYs are resuspended in sterile saline rather than in carbonate buffer. The IgY preparations are delivered by intra-peritoneal injection. The IgY preparations are administered either once, twice or three times a day for a period of 4 days (the treatment period).

The animals are observed for the onset of diarrhea and death during and after the treatment period. The level of protection afforded by each treatment dosage is calculated. If the lowest dose is protective in a significant number of hamsters, then lower doses are tested in subsequent experiments using the above conditions. For example, 1.0 and 0.5 ml of IgY preparation per animal per day for 4 days would be tested to determine the lowest intra-peritoneal dosage sufficient for protection. If only very small doses of IgY are needed to confer protection via intra-peritoneal injection, then the IgY would also be delivered via intra-vascular injection to determine whether intra-vascular delivery of the IgY PEG preparations confer protection from C. difficile infection.

EXAMPLE 41 Treatment of Hamsters Infected with C. difficile using Enteric-Coated IgYs Against a Recombinant C. difficile Toxin A Protein

To determine whether the enterically-coated anti-recombinant toxin A IgY (Example 37a) is effective in treating C. difficile infection in hamsters at a lower dose required using the same IgY without an enteric coating, the following experiment is performed.

The hamster infection model is carried out exactly as described in Example 32c with the exception that enterically-coated antitoxin (Eudragit® L30D-coated pMA1870-2680 which had first be applied to a non-pariel) is used in place of the non-coated IgY in carbonate buffer. Briefly, three groups of hamsters (Sasco) containing 7 members per group are predisposition to infection with Clindamycin-phosphate (Biomol) at 1 mg/100 gram body weight. Twenty-four later, each animal is orally challenged, using an 18 gauge feeding needle, with 1 ml of C. difficile (ATCC 43596) containing approximately 1×10³ organisms in sterile saline. The organisms are grown for 48 hours on CCFA plates (BBL) prior to infection.

Three hours after infection (Day 1), treatment is intimated by oral administration of various concentrations of Eudragit®-coated anti-toxin A IgY as follows. Each group receives 0 (the control group), 2, 20, 50, 100 or 600 mg of enterically-coated IgY once per day for a period of 4 days. The enterically-coated particles are administered orally to the hamsters by placing each dose in a microcentrifuge tube, resuspending the particles in a low pH buffer such as acetate, pH 4.0 (low pH buffers are used to prevent the release of the IgY from the enterically-coated particle prior to delivery to the hamster); the suspension is then orally administered using a 14 gauge feeding needle. The animals are observed for the onset of diarrhea and death during and after the treatment period. The percentage cumulative mortality (i.e., death) and morbidity (i.e., diarrhea) are calculated.

The results form the above experiment (administration of enterically-coated IgY) are compared to the results obtained in Example 32c. In Example 32c, the same infection conditions were employed but the anti-toxin A antibodies were delivered in carbonate buffer and they lacked an enteric coating. In Example 32c, 50% of the hamsters treated after infection with uncoated IgYs were protected from death from C. difficile. The amount of total IgY given per day in Example 32c was about 120 mg. Of that dose, the amount of specific antibody per day necessary achieve that level of protection (i.e., 50% survival) was about 1200 μg of specific IgY. In the present example, the hamsters are each given 2, 20, 50, 100 or 600 mg of enterically coated IgY. Since only 1/5 of the weight of the enterically-coated material is IgY, the actual amount of total IgY administered in the 2, 20, 50, 100 and 600 mg doses is about 0.40 mg, 4 mg, 10 mg, 20 mg and 120 mg, respectively. Of that about 1% is specific anti-recombinant toxin A IgY. The 600 mg dose of the enteric particle (i.e., the Eudragit®-coated anti-recombinant C. difficile toxin A IgY preparation) is roughly equivalent to the amount of antibody delivered in carbonate buffer in Example 32c which gave 50% protection. Comparison of the dose of the enteric particles required to give the same (i.e., 50%) level of protection indicates the degree of increased potency afforded by enterically-coating the IgY preparation. The results of the above experiment demonstrate whether enterically-coated anti-recombinant C. difficile toxin A IgY (Example 37a) is effective in treating C. difficile infection in hamsters at a lower dose as compared to non-coated anti-recombinant toxin A.

Accordingly, the recombinant C. difficile toxin B IgY (i.e., anti-pPB1750-2360) is also enterically-coated using the methods described in Example 37a. The enterically-coated anti-recombinant C. difficile toxin B IgY is tested in the hamster infection model described above alone or in combination with enterically-coated anti-recombinant C. difficile toxin A (i.e., the coated anti-pMA1870-2680 IgY preparation). The results of these experiments demonstrate whether enterically-coated anti-recombinant C. difficile toxin A and B IgYs (Example 37a) are effective in completely protecting animals from the morbidity and mortality associated with C. difficile infection at lower doses as compared to the use of non-coated anti-recombinant C. difficile toxin A and B IgYs.

EXAMPLE 42 Determination of the Minimum Effective Dose of Avian Antibodies in Carbonate Buffer Against Recombinant C. difficile Toxin A and Toxin B to Treat C. difficile-Infected Hamsters

The minimum effective dose of avian antibodies (IgY) raised against recombinant toxin A protein and recombinant toxin B protein necessary to treat C. difficile-associated disease (CDAD) in hamsters was determined. The experiment involved an in vivo infection study in which hamsters were treated with different concentrations of IgYs raised against recombinant toxin A and recombinant toxin B.

Antibodies were generated against recombinant C. difficile toxin A (pMA1870-2680; Interval A-6) using RIBI adjuvant and against the recombinant C. difficile toxin B (pPB1750-2360; Interval B-3) using Freund's adjuvant. The immunization protocol used for each adjuvant was previously described in Example 35. Antibodies were PEG-purified and resuspended at an 8× concentration (all contained about 40 mg/ml IgY) in 0.1M carbonate buffer, pH 9.5.

The infection study involved the testing of three experimental groups (Group I, Group II, and Group III). Group I animals received 2 mls pre-immune IgY. Group II animals received 1 ml of a mixture containing anti-C. difficile toxin A and anti-C. difficile toxin B IgY. Group III animals received 2 ml of the mixture given to Group II. Each group consisted of eight Golden Syrian hamsters (Sasco) weighing an average of 79+/-3.2 gm. The hamsters were housed two or three per cage and were given food and water ad libitum throughout the study. The infection study was performed using the protocol described in Example 31c.

Hamsters were predisposed to infection with C. difficile by I.P. administration of 1 mg/100 gm body weight of Clindamycin-phosphate (Biomol) in 1 ml of sterile water. The Clindamycin was administered I.P. using a 1 ml 27-gauge tuberculin syringe (Terumo). About 24 hours later, the hamsters were each infected orally with approximately 1 ml of sterile saline containing 1×10⁴ C. difficile (strain ATCC 43596). The C. difficile were grown for about 48 hours on CCFA (cycloserine-cefoxitin-fructose-egg yolk agar, a C. difficile-selective and differential medium) plates (BBL) prior to inoculation.

Eight hours after inoculation (Day 1), treatment was initiated. The hamsters in each of the three groups orally received one of three treatments through an 18-gauge feeding needle (Popper). Group I received 2 ml of pre-immune IgY (as an 8× PEG preparation), Group II received 1 ml of immune IgY (i.e., a mixture of antibodies generated against pMA1870-2680 Interval A-6! and pPB1750-2360 Interval B-3!), and Group III received 2 ml of immune IgY.

The immune IgY mixture was prepared by mixing an equal volume of an 8× concentrate of IgY raised against pMA1870-2680 and an equal volume of an 8× concentrate of IgY raised against pPB1750-2360; the resulting mixture was designated A-6/B-3 IgY. The amount of anti-toxin protein specific antibodies contained in this A-6/B-3 IgY mixture was about 1.2 mg/ml of anti-recombinant toxin A IgY and about 400 μg/ml of anti-recombinant toxin B IgY. These amounts were determined by affinity purification as previously described in Example 15c. The amounts of total IgY in the 2 ml IgY dose was about 80 mg, and about 40 mg in the 1 ml dose.

The hamsters were treated once each day for 3 days. The dosing schedule in this treatment regimen differs from that used previous Examples (e.g., Example 32), where the hamsters were treated with 2 ml three times daily for 3 days.! All hamsters were observed for the onset of diarrhea and death during and after the treatment period. The results are shown in FIG. 47 and Table 48.

In FIG. 47, cumulative mortality (expressed as a percentage) is displayed along the ordinate and time (expressed in days) is displayed along the abscissa. The duration of the treatment period, indicated by the horizontal bar in FIG. 47, was 3 days. The administration of Clindamycin and the inoculation with C. difficile ("Infection") is indicated by arrows. The solid black squares represent the Group I hamsters (i.e., hamsters treated with 2 ml of pre-immune IgY). The open squares represent the Group II hamsters (i.e., hamsters treated with 1 ml of the A-6/B-3 IgY). The solid black diamonds represent the Group III hamsters (i.e., hamsters treated with 2 ml of A-6/B-3 IgY).

The results shown in FIG. 47 demonstrate that all the hamsters (i.e., 8/8) in Group III were protected from death. In contrast, only 13% (i.e., 1/8) of the hamsters in both Groups I and II survived. The degree of protection in Group III was statistically significant at P<0.005, using Chi-square analysis.

The following table shows the results observed using A-6/B-3 IgY. The dose given in this table refers to the total (not specific) IgY concentration given to the animals in a 1 or 2 ml dose.

                  TABLE 48                                                         ______________________________________                                         Prevention of Morbidity In Vivo Using A-6/B-3 IgY                              Treatment Group                                                                            % Animals with Diarrhea                                                                         Mean Weight.sup.1                                 ______________________________________                                         Pre-immune, 80 mg                                                                          100              67.3 ± 3.9                                     (Group I)                                                                      A-6/B-3, 40 mg                                                                             100                69 ± 4.2                                     (Group II)                                                                     A-6/B-3, 80 mg                                                                             0                81.6 ± 5.5                                     (Group III)                                                                    ______________________________________                                          .sup.1 Weight expressed in grams ± S.D.; mean starting weight of            hamsters was 79 ± 3.2 gm. For the animals that died, weight was             measured at the time of death. For survivors, the weight was measured          after the end of treatment.                                              

The results shown in Table 48 demonstrate that morbidity was also prevented when the hamsters were treated using 2 ml of A-6/B-3 IgY per day. Morbidity from CDAD was defined here as including diarrhea and weight loss. As shown in Table 48, none of the hamsters in Group III presented with diarrhea. In contrast, all of the hamsters treated with either 1 ml of A-6/B-3 IgY (Group II) or with 2 ml of pre-immune IgY (Group I) presented with diarrhea. Moreover, all but one of the hamsters in Groups I and II died about 48 hours after presenting with diarrhea. The prevention of diarrhea in Group III was statistically significant (P<0.001) in comparison to the other two treatment groups.

In addition, the results shown in Table 48 indicate that hamsters treated with either 2 ml of pre-immune IgY (Group I) or 1 ml of A-6/B-3 IgY (Group II) lost 14% of their mean starting weight prior to infection. In contrast, the animals treated with 2 ml of the A-6/B-3 IgY (Group III) gained about 2% of their mean starting weight at the end of the treatment period.

Based on the above results, the minimal effective therapeutic dose of specific IgY to both recombinant C. difficile toxins A and B (pMA1870-2680 and pPB1750-2360; Intervals A-6 and B-3, respectively) necessary to prevent mortality and morbidity of CDAD in hamsters under the conditions set forth above (i.e., in carbonate buffer) is about 2.4 mg of A-6 IgY and about 800 μg of B-3 IgY per day for 3 days.

EXAMPLE 43 Determination of Specific IgY in the Cecum of a Hamster Treated with Anti-Recombinant C. difficile Toxin A and Anti-Recombinant C. difficile Toxin B

The amount of specific anti-C. difficile toxin IgY found in the cecum of a hamster treated with antibodies raised against the recombinant toxin A (pMA1870-2680; A-6) and the recombinant toxin B (pPB1750-2360; B-3) was determined. The results of this study provide an indication of the approximate therapeutic dose of anti-recombinant toxin A and anti-recombinant toxin B IgY (A-6/B-3 IgY) that must be present at the site of infection to protect an animal infected with C. difficile.

The example involved a) recovery of cecal contents from an untreated hamster and from a hamster treated with anti-recombinant C. difficile toxin A and anti-recombinant C. difficile toxin B IgY (i.e., A-6/B-3 IgY), b) direct determination by ELISA of the amount of specific A-6 IgY and B-3 IgY in the cecal contents, and c) direct determination by ELISA of total IgY in the cecum and indirect determination of specific IgY.

a) Recovery of Cecal Contents from an Untreated Hamster and from a Hamster Treated with Anti-Recombinant C. difficile Toxin A and Anti-Recombinant C. difficile Toxin B IgY (A-6al-3 IgY)

A hamster was treated according to the protocol for Group III animals as described in Example 42 (i.e., a hamster that was successfully treated against CDAD using 2 ml of 8× A-6/B-3 IgY per day for 3 days). Four hours after the last dose of IgY was administered, the hamster was sacrificed using ether. A control hamster was treated with Clindamycin to predispose the animal to infection with C. difficile, but did not receive any IgY nor C. difficile, served as a negative control. The control hamster was sacrificed two days after administration of Clindamycin.

The ceca from both hamsters were removed and most of the cecal contents were collected in a 15 ml centrifuge tube. Each centrifuge tube contained several mls of cecal material. The contents in each tube were vortexed and a 1 ml aliquot from each tube was removed to a 1.5 ml microcentrifuge tube. The microcentrifuge tubes were centrifuged for 1 minute at 14,000 rpm. The resulting supernatant was collected and the specific IgY in the samples was quantitated by ELISA.

b) Direct Determination by ELISA of the Amount of Specific A-6 IgY and B-3 IgY in the Cecal Contents The levels of anti-recombinant toxin A and toxin B IgY present in the cecal contents were detected by ELISA using the protocol described in Example 13c with the following modifications. The 96-well microtiter plates were coated (100 μl/well) overnight at 4° C. with 0.05 μg/ml of recombinant C. difficile toxin A protein pPA1870-2680 (N/C) (Example 29)! or with 1 μg/ml of recombinant C. difficile toxin B protein pPB1750-2360 (Example 18b)! in PBS, pH 7.4. Cecal samples were initially diluted two-fold into PBS and placed in the wells. The diluted samples were then serially diluted 5-fold in the microtiter wells. All samples were tested in duplicate.

Quantitation of specific IgY levels in the cecal samples was determined by comparing the antibody reactivity directed against either recombinant toxin A or recombinant toxin B to the reactivity generated using known amounts of affinity-purified IgY in ELISA assays. IgY specific for recombinant C. difficile toxins A and B were affinity purified as described in Example 15c. Briefly, affinity-purified antibodies were isolated from PEG-purified IgYs from the eggs of hens immunized with either pMA1870-2680 (Interval A-6) or pPB1750-2360 (Interval B-3). The PEG-purified anti-pMA1870-2680 IgY was affinity-purified using a column comprising pMA1870-2680 bound to Actigel (Sterogene). The PEG-purified anti-pPB1750-2360 IgY was affinity purified using an Actigel column containing pPB1850-2360 (Example 15c), a recombinant C. difficile toxin B protein that is 100 amino acids smaller than pPB1750-2360.

The affinity-purified anti-pMA1870-2680 IgY (A-6) and the affinity-purified anti-pPB1750-2360 IgY (B-3) were quantitated by measuring the absorbance at 280 nm and each preparation was diluted to a concentration of 10 μg/ml in PBS. The affinity-purified IgYs were tested by ELISA starting with an initial concentration of 10 μg/ml and at serial 5-fold dilutions in the respective coated-microtiter plate along side the cecal extracts. Rabbit anti-chicken IgY conjugated with alkaline phosphatase (Sigma) was used as the secondary antibody at a dilution of 1:750 to detect the bound IgYs in the ELISA.

Comparison of the ELISA reactivity equivalence between the cecal extracts to the known concentrations of the affinity-purified IgY allowed the amount of specific anti-recombinant IgY present in the cecal extracts to be estimated. From the ELISA results the amount of specific anti-pMA1870-2680 IgY (A-6) was found to be about 12 μg/ml. The amount of specific anti-pPB1750-2360 IgY (B-3) was determined to be about 800 ng/ml. These concentrations of specific IgYs provide estimates of the effective therapeutic concentrations necessary to achieve protection at the site of infection. Since the amount of specific IgY given orally prior to collection of the cecal fluid was about 2400-2800 μgs against the toxin A recombinant and from 400-800 μgs against the toxin B recombinant, there was approximately a 200-233 fold reduction and 500-1000 fold reduction in the detectable amount of anti-recombinant toxin protein IgY found in the cecum directed against Interval A-6 and Interval B-3, respectively.

These results were obtained using antibodies present in carbonate buffer. If the orally administered anti-recombinant toxin IgY were properly protected from degradation during passage through the GI tract (i.e. through use of an enteric coating as described in Example 37) the effective therapeutic dose required for oral administration would be less than that specified in Example 42.

c) Indirect Determination of the Amount of Specific Anti-Recombinant C. difficile Toxin A and Toxin B IgY in the Cecal Contents by ELISA

In order to confirm the values determined in Example 43(b) for the amount of anti-recombinant toxin IgY levels found in the cecum of a treated hamster, the following experiment was performed. The standard ELISA format was used (described in Example 13c) unless otherwise specified.

The cecal extracts from an untreated hamster (serving as the negative control) and one treated with both anti-recombinant toxin A and anti-recombinant toxin B IgY described in section a) above! were used in this experiment. The total cecal IgY, not just the amount of cecal IgY specific for the recombinant C. difficile toxin proteins, was directly determined. Because the percent amounts of specific antibody contained within the IgY preparations was known, the determination of the total IgY level present in the cecal extracts allowed the amounts of specific antibody in the cecal extract to be calculated.

A sandwich ELISA assay was used to capture IgY in the cecal material as follows. Rabbit anti-chicken IgG (Capped at 0.1 μg/ml in PBS was used to coat a microtiter plate (100 μl per well) overnight at 4° C. Both of the cecal extracts were tested at an initial dilution of 1:500 and at serial 5-fold dilutions to a final dilution of 1:312,500. All sample dilutions were tested in duplicate. Affinity-purified antibodies directed against recombinant toxin A (pPA1870-2680, Interval A-6)were diluted to 0.1 μg/ml and then further diluted serially by five-fold to a final concentration of 0.16 ng/ml, was also tested by ELISA for allow for quantitation by comparison. After incubation and washing, rabbit anti-chicken alkaline phosphatase IgG (Sigma) was added (at 1:1000 dilution) to the plates. The plates were then washed and substrate (p-nitrophenyl phosphate) was added and the plates were evaluated as described in Example 13c.

As described above in Example 43(b), the ELISA reactivity obtained using the affinity purified anti-recombinant toxin A IgY was matched to that ELISA activity generated in dilutions of cecal extract, to quantitate the amount of total IgY found in the cecum of the treated hamster.

From the results of the ELISA assay, the amount of total IgY in the cecum of the treated hamster was estimated to be 50 μg/ml. Affinity purification studies showed that total IgY preparations comprised about 7% or 3.5 μg/ml IgY specific for recombinant toxin A (anti-A-6 IgY) and about 1-2% or 500-1000 ng/ml IgY specific for anti-recombinant toxin B (anti-B-3 IgY). The concentrations of both of the specific IgYs detected here correlates fairly closely with the amounts detected above in Example 43(b), namely, 3.5 μg/ml versus 12 μg/ml for anti-Interval A-6 and 800 ng/ml versus 500-1000 ng/ml for anti-Interval B-3.

The results above and in section b) of this Example, both indicate that very low levels of specific anti-Interval A-6 and B-3 IgY's were detected at the site of the C. difficile infection. Since the hamster was protected from CDAD, this level of anti-recombinant toxin A and B is within the therapeutic range. These results also support the proposition that much lower levels of anti-recombinant toxin IgYs would need to be orally administered if they were delivered using means to prevent degradation in the GI tract (i.e., enteric coating of IgY).

EXAMPLE 44 Treatment of Diarrheic Hamsters Using Anti-Recombinant C. difficile Toxin A Protein IgY

To determine whether hamsters presenting with diarrhea after infection with C. difficile could be effectively treated using the anti-recombinant C. difficile toxin A IgY alone or whether a combination of anti-recombinant toxin A and B IgY is required, the following experiment was performed.

Hamsters were given Clindamycin and infected with C. difficile essentially as described in Example 32c. The anti-recombinant toxin A IgY and anti-recombinant toxin B IgY were produced against pMA1870-2680 (Interval A-6) and pPB1750-2360 (Interval B-3), respectively.

Three groups of hamsters (Sasco) were predisposed to C. difficile infection by I.P. injection of 1 ml of Clindamycin-phosphate (Biomol) at 1 mg/100 g body weight. About 24-hours later, each hamster was challenged, using an 18 gauge feeding needle, with a 1 ml inoculum containing approximately 1×10⁴ C. difficile (ATCC 43596) organisms in sterile saline. The bacteria were grown for about 48 hours on CCFA agar (BBL) plates prior to infection.

The three groups, each containing nine to ten members, were given IgY preparations using a feeding needle. Group I received pre-immune IgY; Group II received anti-recombinant toxin A IgY (anti-A-6 IgY); Group III received a mixture of anti-recombinant toxin A IgY and anti-recombinant toxin B IgY (anti-A-6/B-3 IgY). Each IgY preparation comprised an 8× PEG prep in 0.1M carbonate buffer, pH 9.5 and contained about 40 mg/ml of protein. To generate the anti-A-6/B-3 IgY mixture, equal volumes of the two 8× concentrates (i.e., anti-A-6 and anti-B-3) were mixed together. The amount of specific anti-recombinant C. difficile toxin A IgY in the anti-A-6 IgY preparation was about 2.8 mg/ml. The amount of specific anti-recombinant C. difficile toxin A IgY in the anti-A-6/B-3 IgY prep per ml was therefore half that of the A-6 IgY prep (i.e., 1.4 mg/ml). About 200-400 μg/ml of specific anti-recombinant C. difficile toxin B IgY was present in the anti-A-6/B-3 IgY preparation.

After the onset of diarrhea was detected in each individual hamster, that animal was dosed with 2 ml of their respective treatment preparations (i.e., either pre-immune, anti-A-6 IgY or anti-A-6/B-3 IgY). The onset of diarrhea was detected in the hamsters from 20 to 44 hours post-inoculation with C. difficile. The majority of the hamsters (82%) exhibited diarrhea within 24 hours post-inoculation with the organisms. The majority of the animals were given 3 doses of IgY per day at roughly 4 hour intervals for 2 days; however, some hamsters were only dosed once or twice on the first day of treatment due to a later onset of diarrhea.

The results of this experiment indicated that the anti-A-6 IgY was able to protect many of the hamsters from death, even if given after the onset of diarrhea. About half (55%) of the hamsters treated with anti-A-6 IgY survived for approximately one month, while only 11% of the hamsters treated with preimmune IgY and 20% of the hamsters treated with anti-A-6/B-3 IgY survived.

Because it appears that the anti-A-6 IgY is the most important component for prevention of death in the hamster model (e.g., Example 32(a)), the results obtained in hamsters treated with the anti-A-6/B-3 IgY (which contains half the amount of specific anti-A-6 IgY compared to the A-6 IgY alone preparation) was not unexpected (only 20% of the animals were protected from death). In this Example, anti-A-6 IgY alone could not prevent mortality in 50% of the hamsters, and anti B-3 IgY alone did not provide protection. In addition, the results obtained in previous studies (e.g., Example 32c) indicated that the anti-B-3 antibodies are more important in preventing the onset of diarrhea rather than in preventing death due to CDAD.

All of the animals that were successfully treated with the anti-A-6 IgY exhibited mild diarrhea before treatment was started. If diarrhea was severe and neurological symptoms were present before treatment was initiated, the hamsters could not be successfully treated with oral anti-A-6 IgY.

The above hamster treatment experiment was repeated, with the exception that only pre-immune or anti-A-6 IgY were administered. Ten to eleven hamsters per group were treated with 2 ml of 8× IgY concentrates after diarrhea has been detected in each individual hamster. The treatment schedule was as described above.

Diarrhea was detected in the hamsters from about 20 to 45 hours after inoculation with C. difficile. Eighty-five percent of the animals (17/20) presented with diarrhea about 28 hours after infection. The data from these two studies were combined and are shown in FIG. 48.

In FIG. 48, the percentage cumulative mortality is displayed along the ordinate and the time (in days) is displayed along the abscissa. The treatment period is indicated by the use of the bar between days 2 and 3. The administration of Clindamycin and the inoculation with C. difficile organisms (marked as "Infection" in FIG. 48) is indicated by the arrows. The solid black squares represent hamsters which received pre-immune IgY and the open squares represent hamsters which received anti-A-6 IgY.

The data shown in FIG. 48 demonstrates that 45% of the hamsters treated with anti-A-6 IgY post-diarrhea survived after treatment (diarrhea resolved itself in most of these hamsters). The results obtained using anti-A-6 IgY as compared to the results obtained using pre-immune IgY was statistically significant at P<0.05. All of the hamsters treated with anti-A-6 IgY survived long term (>1 month after treatment until the termination of the experiment).

These results provide the first description of a treatment regime which can be used to treat hamsters after the onset of diarrhea due to infection with C. difficile. Furthermore, these results demonstrate that the anti-recombinant C. difficile toxin A IgY is an effective therapeutic even when administered at the late stages of CDAD, (i.e.,after the onset of diarrhea).

EXAMPLE 45 Treatment of Established C. difficile Infection Using Anti-Recombinant C. difficile Toxin A Protein and Toxin B Protein IgYs Generated Using Two Different Adjuvants.

The ability of recombinant toxin A and toxin B proteins produced using the pET vector to elicit neutralizing IgY in the hens capable of protecting hamsters against C. difficile infection was examined. In previous studies (Example 32 or Example 44), the IgYs were generated against recombinant C. difficile toxin A proteins expressed using the pMal vector (pMA1870-2680, Interval A-6). Because recombinant proteins expressed using the pET system could be isolated in a more highly purified form, as compared to proteins expressed using the pMal vector, production of antibodies against the toxin A recombinant produced in pET (pPA1870-2680, A-6) was preferred.

The anti-pPA1870-2680 IgYs were tested in the hamster model along with antibodies raised against the toxin B protein also expressed using the pET vector (pPB1750-2360, Interval B-3). The use of a common expression system to produce both recombinant toxins has definite manufacturing advantages. For example, the same affinity-purification columns and protocols can be used for both recombinants and both antigens should be of comparable purity and yield.

A further objective of this example was to generate antibodies against both pET produced A-6 and B-3 toxin proteins using the same adjuvant. In previous examples (Example 32 or Example 44), the IgYs tested were generated against either A-6 or B-3 recombinants using different adjuvants (the RIBI adjuvant was used for the anti-recombinant toxin A IgY and Freund's adjuvant was used for the recombinant toxin B IgY).

The example involved a) the immunization of hens with recombinant C. difficile toxin proteins expressed using the pET vector and 2 different adjuvants and the determination of anti-recombinant protein IgY titers by ELISA and b) treatment of C. difficile-infected hamsters using a mixture of Gerbu or Quil A-generated anti-recombinant toxin A (A-6) and toxin B (B-3) IgY.

a) Immunization of Hens with Recombinant C. difficile Toxin Proteins Expressed Using the pET Vector and Two Different Adjuvants and the Determination of Anti-Recombinant Protein IgY Titers by ELISA

Hens were immunized with recombinant proteins expressed using the pET vector; nickel column affinity-purified recombinant toxin A (pPA1870-2680) or the recombinant toxin B (pPB1750-2360) proteins were mixed with either the Quil A (Accurate Scientific) or Gerbu (CC Biotech) adjuvants. These two adjuvants were chosen on the basis of performance (shown in Example 35) and cost. The immunization protocol followed was basically that described in Example 35.

Briefly, hens were immunized with 100 μg of pPA1870-2680 for the first four immunizations followed by two immunizations using 1 mg of protein. Hens were immunized six times using 1 mg of pPB1750-2360 per immunization. Five hundred microliters of a solution containing either recombinant toxin protein and either 5 μg of Gerbu or 75 μg of Quil A was administered sub-cutaneously to each hen. About 1 week after the last boost, the eggs were collected and the IgYs in each group (four groups of hens, using both toxin recombinants with both adjuvants) were extracted using PEG as described in Example 1. IgY from preimmune eggs was also processed.

The IgYs were resuspended in 0.1M carbonate buffer, pH 9.5 at 8× yolk concentration (about 40 mg/ml) and an ELISA was performed (as described in Example 35) to determine the anti-recombinant toxin A and anti-recombinant toxin B titers. The antibody titers generated against either the pPA1870-2680 (A-6) or pPB1750-2360 (B-3) proteins using either the Gerbu or Quil A adjuvants was found to be 1:62,500. By affinity purification, the amount of specific A-6 and B-3 IgY using Gerbu was 4.3% and 1.0% respectively. The amount of specific IgY using Quil A was 2.2% for A-6 and 1.9% for B-3.

b) Treatment of C. difficile-Infected Hamsters Using a Mixture of Gerbu or Quil A-Generated Anti-Recombinant Toxin A (A-6) and Toxin B (B-3) IgY

Equal volumes of the anti-A-6 and anti-B-3 IgY PEG preps generated in section a) using the same adjuvant were mixed. These preparations were designated A-6/B-3 Gerbu and A-6/B-3 Quil A. The hamster treatment study was performed exactly as described in Example 42. Six hours after challenge with 10⁴ C. difficile organisms (ATCC strain 43596), the hamsters were treated with 2 ml of either pre-immune IgY or an immune IgY preparation (A-6/B-3 Gerbu and A-6/B-3 Quil A). The hamsters were treated with 2 ml of IgY for two more days, once per day.

The results of this hamster treatment study are shown in FIG. 49. In FIG. 49, the percentage cumulative mortality is displayed along the ordinate and the time (in days) is displayed along the abscissa. The treatment period is indicated by the use of the bar between days 1 and 3. The administration of Clindamycin and the inoculation with C. difficile organisms (marked as "Infection" in FIG. 49) is indicated by the arrows. The solid black squares represent hamsters which received pre-immune IgY; the open squares represent hamsters which received anti-A-6/B-3 Gerbu IgY and the solid black diamonds represent hamsters which received anti-A-6/B-3 Quil A IgY. The results shown in FIG. 49 demonstrate that both immune IgY preparations (A-6/B-3 Gerbu and A-6/B-3 Quil A) completely protected the hamsters from death due to CDAD. Nine out of nine of the hamsters treated with either of the immune IgY preparations survived infection with C. difficile, while all nine of the hamsters treated with pre-immune IgY died. The survival rates seen using either the A-6/B-3 Gerbu or A-6/B-3 Quil A preparations were statistically significant compared to the result obtained using pre-immune IgY (P value of <0.001 using Chi-square analysis).

Three out of nine of the animals treated with A-6/B-3 Gerbu and one out of nine of the animals treated with A-6/B-3 Quil A presented with very slight diarrhea. The slight diarrhea seen in these treated hamsters (compared to the total absence of diarrhea seen in previous Examples, such as Example 32) may be due to the lower antibody titer of the preparations used here (1:62,500 versus 1:125,000). Additional booster immunizations should increase titers to the 1:125,000 range for hens immunized using either Gerbu or Quil A adjuvants, and thus increase therapeutic potency against diarrhea.

The above results indicate that the recombinant C. difficile toxin A and B proteins can be produced using the pET vector (in place of the pMal vector) without deleterious effects upon the production of neutralizing antitoxin. Furthermore, the same adjuvant can be used in conjunction with the pET-produced proteins to elicit in vivo neutralizing IgY. Moreover, the same adjuvant can be used with both recombinant toxin proteins to produce a therapeutic anti-recombinant IgY response in vivo.

EXAMPLE 46 Production of Antibodies Using a Mixture Containing Recombinant C. difficile Toxins A and B in Hens

The ability to raise chicken antibodies directed against both a recombinant C. difficile toxin A and toxin B protein using a mixture of both proteins as a combined immunogen was investigated. The example involved a) immunization of hens with a mixture of recombinant C. difficile toxin A and B proteins and b) purification and detection of anti-recombinant C. difficile toxin A and toxin B IgY.

a) Immunization of Hens With a Mixture of Recombinant C. difficile Toxin A and B Proteins

Egg-laying Leghorn hens were immunized with a mixture of recombinant toxin A (pPA1870-2680, Interval A-6) and recombinant toxin B (pPB1750-2360, Interval B-3) proteins; both recombinant proteins were expressed using the pET vector system. Two groups of hens (each containing 4 hens) were immunized with 500 μg of each recombinant protein mixed with either Quil A (Accurate Scientific) or Gerbu (CC Biotech) adjuvant. A total volume of 1 ml containing the recombinant proteins and either 5 μg Gerbu or 75 μg Quil A adjuvant was administered to each hen. The immunization protocol followed for each adjuvant was as described in Example 35. The hens were immunized twice 2 weeks apart.

b) Purification and Detection of Anti-Recombinant C. difficile Toxin A and Toxin B IgY

About 1 week after the last boost, 3 eggs from each group were collected and IgY was extracted using PEG as described in Example 1. The IgYs were resuspended in PBS (pH 7.4) at a 4× concentration each containing about 20 mg/ml total protein. Preimmune IgY served as a negative control.

The amount of anti-recombinant toxin A (A-6 IgY) and anti-recombinant toxin B (B-3 IgY) antibodies present in the two immune IgY preparations was determined by ELISA essentially as described in Example 13c or Example 43b. Briefly, the wells of a microtiter plate were passively coated with either the toxin A recombinant (pPA1870-2680) or the toxin B recombinant (pPB1750-2360). The IgY samples were initially diluted 250-fold, then serially diluted 5-fold. All samples were tested in duplicate. Rabbit anti-chicken IgG alkaline phosphatase (Sigma) diluted 1:1000 was used to detect the specific IgY.

The results of these ELISA assays revealed that both recombinant toxin antigens were able to elicit an IgY response in the hen. The antibody titers are expressed as the reciprocal of the highest dilution that was found to be about 3-fold higher in ELISA reactivity compared to pre-immune (i.e., the negative control) at the same dilution. The titers for both the anti-A-6 IgY and anti-B-3 IgY generated using the Gerbu adjuvant were very low at about 1:250. The titer for the anti-A-6 IgY and anti-B-3 IgY generated using the Quil A adjuvant was from 5 and 25 fold-higher compared to the Gerbu adjuvant. Antibody titers generated using Quil A for the anti-A-6 IgY was greater than 1:6250 and greater than 1:1250 for the anti-B-3 IgY.

While the IgY titers against the recombinant toxins using Quil A are lower than the levels achieved previous Examples (e.g., Example 32) (mainly because these hens in this example have been immunized only twice; in comparison the hens in previous examples were immunized 5 to 10 or more times and the resulting anti-toxin protein titers reached 1:100,000 or more), the results indicated that both recombinant toxin proteins appear to be equally antigenic in the hens and the antibodies produced to each were present at comparable levels. The results also indicated that the level of the antibody response generated depends on the adjuvant used. It was found that the Quil A adjuvant invoked a higher anti-A-6 and anti-B-3 IgY response early during the immunization process in comparison to the results obtained using the Gerbu adjuvant.

EXAMPLE 47 Affinity Purification of Native C. difficile Toxin A Using Anti-Recombinant C. difficile Toxin A Antibodies

Avian antibodies (IgY) raised against recombinant C. difficile toxin A protein were affinity purified using interval A-6 as the affinity ligand. The resulting specific antibodies were then immobilized on a solid support to purify native toxin A from C. difficile (ATCC #43255) organisms grown in dialysis bags submerged in BHI broth. The following example describes the a) affinity purification of avian antibodies directed against a recombinant fragment of toxin A and generation of a toxin A affinity column, b) growth of C. difficile organisms to produce toxin A and B in dialysis bag culture supernatants, c) affinity purification of toxin A, d) in vitro characterization of affinity purified C. difficile toxin A, e) investigation of an alternate strategy for coupling the anti-A-6 IgY to a solid support to affinity purify toxin A, f) affinity purification of C. difficile toxin A on affinity column generated by periodate oxidation of A-6 IgY and g) in vitro characterization of affinity purified C. difficile toxin A.

a) Affinity Purification of Avian Antibodies Directed Against a Recombinant Fragment of Toxin A and Generation of a Toxin A Affinity Column

Antibodies specific for Interval A-6 (aa 1870-2680) of C. difficile toxin A were affinity purified to provide reagents for the generation of an affinity column to permit purification of C. difficile toxin A from liquid culture supernatants and to provide an immunoassay reagent to permit detection of C. difficile toxin A in culture supernatant and affinity purified C. difficile toxin A samples.

i) Affinity purification of A-6 IgY

Hyperimmune IgY from eggs containing antibodies to A-6 recombinant protein using Freund's adjuvant was extracted using the PEG fraction method (Example 1). The antibody-containing supernatant was applied to a A-6 affinity column, made by covalently coupling pPA1870-2680 protein (prepared in Example 29) to Actigel A affinity resin (Sterogene Biochemicals) according to manufacturer's instructions. Approximately 10.2 mg of pPA1870-268 (A-6) protein was coupled to 5 ml Actigel affinity resin. The anti-A-6 IgY was eluted with Actisep elution media (Sterogene Biochemicals) as described in Example 15c, and dialyzed against PBS for 24-48 hours at 2°-8° C.

ii) Coupling of Affinity-Purified Anti-A-6 IgY to an Activated Affinity Resin to Make a C. difficile Toxin A Affinity Column

An initial toxin A affinity column was prepared as described in Example 48a below, by coupling the anti-A-6 IgY to Actigel A affinity resin. By comparing the pre- and post-coupling absorbance values of the IgY at 280 nm, it was estimated that 58%, or about 7.6 mg, of the anti-A-6 IgY was coupled to the affinity resin.

b) Growth of C. difficile Organisms to Produce Toxins A and B in Dialysis Bag Culture

C. difficile strain #43255 was grown as described in Example 49b, sections iv and v, below. SDS-PAGE/Western blot analysis was conducted to evaluate the C. difficile dialysis bag culture supernatants for the presence of toxin A as follows.

The dialysis bag culture supernatant samples and a known toxin A sample purchased commercially were analyzed as described in Example 49b, section iv, with the exception that affinity purified A-6 IgY was used as the primary antibody for the western blot.

Following SDS-PAGE (5% polyacrylamide gel) the proteins were transferred to nitrocellulose using a Milliblot transfer apparatus (Millipore) according to the manufacturer's instructions. The blot was temporarily stained with 10% Ponceau S, and blocked overnight in PBS containing 1 mg/ml dry milk. The preimmune and anti-A-6 IgY primary antibodies were diluted to 1 μg/ml in PBS containing 1 mg/ml BSA, and the appropriate antibody was incubated with the corresponding blot for 2 hours at room temperature with gentle agitation. The strips were washed with PBS (10 mM sodium phosphate, 150 mM NaCl, pH 7.2), BBS-Tween (0.1M boric acid, 0.025M sodium borate, 1M NaCl, 0.1% v/v Tween 20) and PBS to remove unbound primary antibody, and incubated with Rabbit anti-chicken IgG alkaline phosphatase conjugated secondary antibody (Sigma Chemical Co), diluted 1:2000 in PBS/BSA. The blots were washed to remove unbound secondary antibody, and the strips were developed in BCIP/NBT substrate solution (as described in Example 48 below).

Both culture supernatant samples analyzed appeared to contain immunoreactive C. difficile toxin A when analyzed by Western blot. This protein co-migrated with the commercial toxin A and was recognized by the affinity-purified anti-A-6 IgY. The culture supernatant samples were pooled prior to affinity purification of toxin A. The pooled culture supernatants were not concentrated prior to loading on the affinity column.

c) Affinity Purification of C. difficile Toxin A

The C. difficile toxin culture supernatant samples were affinity purified as described in Example 48c. The volume of the Actisep fraction following elution and dialysis was 42 ml, 15 ml of which were removed and concentrated to 3 ml prior to analysis. A Centricon 30 concentrator (Amicon) was used to concentrate the sample.

d) Analysis of C. difficile Culture Supernatant, Actisep-Eluted Fraction and Column Effluent for the Presence of Toxin A

In order to determine the presence or absence of toxin A in the Actisep eluted sample and effluent from the affinity column, these samples were analyzed by SDS-PAGE and Western blot along with the culture supernatant starting material. These analyses were performed as described in section b above to evaluate the relative amount of toxin A in the samples and the efficiency of the affinity purification.

The resulting Western blot is shown in FIG. 50. In FIG. 50, lanes 1-3 were incubated with pre-immune IgY as the primary antibody and lanes 4-6 were incubated with anti-A-6 IgY as the primary antibody. Lanes 1 and 4 contain culture supernatant starting material; lanes 2 and 5 contain column flow-through and lanes 3 and 6 contain affinity purified toxin A.

The results shown in FIG. 50 demonstrated that immunoreactive toxin A was detected in the culture supernatant starting material and the Actisep fraction. Furthermore, no toxin A was observed in the column effluent sample, indicating most of the toxin was bound by the affinity column. There appeared to be significantly more toxin A in the starting material than in the Actisep fraction. Since the column effluent apparently contains no toxin A, the difference in toxin A amounts between the starting material and Actisep fraction suggested a significant amount of the toxin was still bound to column, even after Actisep elution. One possible explanation for the inability of the Actisep to elute all of the toxin A is the tendency for toxin A to bind nonspecifically to the carbohydrate region of molecules such as immunoglobulins. This is possible because the anti-A-6 IgY on the column is coupled via primary amines, which would allow for a subpopulation of the IgY to couple via the Fab region, leaving the carbohydrate-containing Fc region accessible for binding to toxin A.

e) Investigation of an Alternate Strategy for Coupling the Anti-A-6 IgY to a Solid Support to Affinity Purify Toxin A

The possibility of coupling IgY to a solid support by periodate oxidation of the carbohydrate region was next examined. This method of coupling was predicted to accomplish the following: 1) couple the IgY to the support via the Fc region, leaving the Fab regions accessible for binding to C. difficile toxin, and 2) alter the carbohydrates enough to eliminate or reduce the nonspecific binding of C. difficile toxin A.

i) Oxidation of Anti-A-6 IgY with Sodium Periodate

The anti-A-6 IgY was oxidized using sodium periodate (Sigma Chemical Co) as follows. A sodium periodate stock solution was made by dissolving 25 mg of sodium periodate in 1.2 ml of distilled, deionized water. To six ml of A-6 IgY (at 2.7 mg/ml) in a 15 ml polystyrene tube 600 μl (0.1 volume) of the sodium periodate stock solution was added. The tube was then covered with aluminum foil and mixed gently at room temperature for 1 hour and 20 minutes. Glycerol was then added to a final concentration of 20 mM, and the tube was inverted for 10 more minutes. The solution was then dialyzed against 100 mM sodium acetate, 150 mM NaCl, pH 5.5 to remove the sodium periodate.

ii) Coupling of Oxidized IgG to Affi-Gel Hz Hydrazide Gel (BioRad)

Six ml of Affi-Gel resin was washed with coupling buffer (100 mM sodium acetate, pH 5.5 and 150 mM sodium chloride). The oxidized anti-A-6 IgY was filtered through a glass fiber syringe filter to remove the precipitate which had formed during the oxidation procedure, and a 100 μl aliquot was removed for A₂₈₀ analysis. The washed Affi-Gel resin was added to the oxidized IgY in a 15 ml polystyrene tube, and the tube was inverted overnight at room temperature (Total volume=12 ml).

iii) Determination of Coupling Efficiency

The anti-A-6 IgY-Affi-Gel affinity resin was poured into a BioRad Econo column and the unbound antibody was washed through the resin and saved for A₂₈₀ analysis. The resin was then washed with 1 bed volume of PBS (10 mM sodium phosphate, 0.5M NaCl, pH 7.2). This wash was also collected and saved for A₂₈₀ analysis. The resin was then washed with several more volumes of PBS, and treated with the Actisep elution buffer (Sterogene Bioseparations) to ensure no unbound antibody remained in the resin. By comparing the pre-and post-coupling A₂₈₀ values of the A-6 IgY, it was estimated that 95%, or 8.4 mg, of the IgY was coupled to the resin.

f) Affinity Purification of C. difficile toxin A on Affinity Column Generated by Periodate Oxidation of Anti-A-6 IgY

Two dialysis bag culture supernatants, grown as described in Example 48b, sections iv and v, were pooled and concentrated to about 10.5 ml using an Amicon centriprep concentrator. The pooled, concentrated supernatants were then applied to the anti-A-6 IgY Affi Gel affinity column and the column effluent was collected and reloaded several times to bind as much toxin as possible. The unbound protein was then removed by washing the column with several bed volumes of PBS and the bound toxin A was eluted with 2 bed volumes of Actisep elution media. The column effluent was saved for analysis to evaluate the efficiency of the affinity purification. The Actisep-eluted toxin was then dialyzed against TBS for 24-48 hours at 2°-8° C., and concentrated from 53 to 3 ml using a Centriprep concentrator (Amicon).

g) In Vitro Characterization of Affinity Purified C. difficile Toxin A

i) Protein Assay

The purified toxin concentration was determined using a BCA protein assay (Pierce) and was found to be 70 μg/ml, or about 210 μg total from 37 ml of culture supernatant, indicating there was about 5.7 μg of toxin/ml of culture supernatant.

ii) Comparison of Toxin Purity and Retention Times by HPLC

HPLC analysis was used to compare both the purity and retention times of the affinity purified toxin A samples. Commercial and affinity purified toxin A samples were applied to a Shodex KW 803 HPLC column and eluted with PBS, using a Waters HPLC system. The toxin A retention times were approximately 7 minutes for both toxin samples, suggesting the toxins are identical. Furthermore, the purities of both toxins were similar.

iii) Western Blot Analysis of Culture Supernatant Starting Material, Affinity Purified Toxin A and Column Effluent (flow through)

In order to evaluate the efficiency of the affinity purification and immunochemically identify the affinity purified toxin A, the culture supernatant, affinity purified toxin A, and column effluent samples were electrophoresed by SDS-PAGE on a 5% gel under reducing conditions and transferred to nitrocellulose using standard methods. The blot was temporarily stained with 10% Ponceau S to allow the lanes to be marked and the remaining protein binding sites were blocked overnight at 2°-8° C. with a PBS solution containing 1 mg/ml dry milk. The blot was cut into two halves, one of which was incubated with anti-A-6 IgY primary antibody, diluted to 1 μg/ml in PBS containing 1 mg/ml BSA, and the second half incubated with preimmune IgY diluted to 1 μg/ml in PBS/BSA. After a two hour incubation in the presence of the primary antibody (with gentle agitation), the unbound primary antibody was removed with successive washes of PBS, BBS-Tween and PBS. Rabbit anti-chicken IgY alkaline phosphatase conjugated secondary antibody, diluted 1:2000 in PBS containing 1 mg/ml BSA was then added to each blot. After two hours, the blots were washed to remove unbound secondary antibody and developed with BLIP/NBT (Kirkegaard and Perry) substrate solution. Color development was stopped by flooding the blots with water. The resulting Western blot is shown in FIG. 51.

In FIG. 51, lanes 1-7 were incubated with anti-A-6 IgY as the primary antibody and lanes 8-15 were incubated with pre-immune IgY as the primary antibody. Lanes 1 and 9 contain broad range molecular weight markers (BioRad). Lanes 2 and 10 contain C. difficile culture supernatant #1. Lanes 3 and 11 contain C. difficile culture supernatant #2. Lanes 4 and 12 contain C. difficile culture supernatants #1 and #2 (pooled). Lanes 5 and 13 contain column flow-through. Lanes 6 and 14 contain affinity purified Toxin A (high load; i.e., 2× the load shown in lanes 7 and 15). Lanes 7 and 15 contain affinity purified Toxin A (low load). Lane 8 does not contain any sample material (blank).

The affinity purified toxin A sample (lane 7) was 3.5 fold more concentrated than the pooled starting material sample (lane 4); however, 1/3 the volume (5 μl vs 15 μl) of the affinity purified sample was loaded compared to the pooled starting material sample. Consequently, if most of the toxin A was recovered from the column, the toxin A levels detected on the Western blot should be similar. As shown in FIG. 51, the signals corresponding to the main high molecular weight bands are comparable. Therefore, the recovery of toxin A from the affinity column appeared to be quantitative.

EXAMPLE 48 Affinity Purification of Native C. difficile Toxin B Using Anti-Recombinant C. difficile Toxin B Antibodies

Arian antibodies (IgY) raised against recombinant C. difficile toxin B protein (pPB 1750-2360; Interval B-3) were affinity purified using Interval B-3 (i.e., aa 1750-2360 of C. difficile toxin B) as the affinity ligand. The resulting purified anti-Interval B-3 specific antibodies were then immobilized on a solid support to facilitate purification of native toxin B derived from C. difficile organisms (ATCC #43255) grown under conditions favorable for toxin production.

The example involved a) affinity purification of avian antibodies directed against a recombinant fragment of C. difficile toxin B and generation of a C. difficile toxin B affinity column, b) growth of C. difficile organisms to produce toxins A and B in liquid culture and dialysis bag culture supernatants, c) affinity purification of C. difficile toxin B, and d) in vitro and in vivo characterization of affinity purified toxin B from C. difficile.

a) Affinity Purification of Avian Antibodies Directed Against a Recombinant Fragment of C. difficile Toxin B and Generation of a C. difficile Toxin B Affinity Column

Antibodies specific for Interval B-3 of C. difficile toxin B protein were affinity purified to provide reagents for the generation of an affinity column to permit purification of C. difficile toxin B from liquid culture supernatants and to provide an immunoassay reagent to permit detection of C. difficile toxin B in culture supernatants and affinity-purified C. difficile toxin B samples.

i) Affinity Purification of Anti-Interval B-3 IgY

Hyperimmune IgY was extracted from eggs containing antibodies to the Interval B-3 recombinant protein (pPB 1750-2360) generated using Gerbu adjuvant (see Example 45) using the PEG fractionation method (Example 1). The antibody-containing supernatant was applied to an Interval B-3 affinity column, made by covalently coupling pPB 1750-2360 protein (prepared in Example 29) to Actigel A affinity resin (Sterogene) as described in Example 15c. This fragment was chosen because it contains the C. difficile toxin B repeat region and does not contain regions of homology with the C. difficile toxin A protein, therefore the resulting purified antibody should not cross-react with C. difficile toxin A. The anti-Interval B-3 antibodies (anti-B-3 IgY) were eluted from the column with 4M guanidine HCl, pH 8.0 and dialyzed against PBS for 24 to 48 hours at 2°-8° C.

ii) Coupling of Affinity-Purified Anti-B-3 IgY to an Activated Affinity Resin to Make a C. difficile Toxin B Affinity Column.

A C. difficile toxin B affinity column was made by coupling 11 mg of the affinity purified avian anti-B-3 antibodies prepared above to 5 ml of Actigel affinity resin (Sterogene). A coupling time of 30 minutes was used rather than the minimum 2 hours recommended by the manufacturer in order to minimize the number of sites where each antibody molecule is coupled to the resin, thereby making the antibody more accessible to the toxin. In addition, the column was only exposed to high salt buffers or the Actisep elution buffer (Sterogene); no guanidine solutions were utilized during the preparation of the affinity column in order to minimize denaturation of the anti-B-3 antibodies. Comparison of the pre- and post-coupling absorbance values of the IgY at 289 nm, revealed that approximately estimate 62%, or about 6.8 mg, of the anti-B-3 IgY was coupled to the resin.

b) Growth of C. difficile Organisms to Produce Toxins A and B in Liquid Culture and Dialysis Bag Culture Supernatants

i) Liquid Culture of C. difficile in BHI Broth

A frozen stock vial of C. difficile (ATCC #43255) was thawed and plated on CCFA plates (BBL) and grown for 36 to 48 hours at 37° C. in an anaerobic chamber. Colonies were harvested from the CCFA plates using a sterile swab. The harvested colonies were used to inoculate a 20 ml liquid culture of BHI broth (BBL). This culture was grown for approximately 24 hours in an anaerobic jar at 37° C. Ten milliliters of the overnight culture were used to inoculate a 500 ml liquid culture of BHI broth, and the culture was grown for approximately 72 hours at 37° C. in an anaerobic chamber.

ii) Harvest of Liquid Culture Supernatant

The 72 hour culture was centrifuged at 5000 rpm (4420×g) for 10 minutes in a Beckman J2-21 centrifuge to pellet the C. difficile organisms and the supernatant was filtered through a 0.45μ filter (Nalgene) and saved for toxin purification and analysis at 2°-8° C.

iii) In Vitro Analysis of Culture Supernatant to Detect Presence of C. difficile Toxin B

In order to determine whether C. difficile toxin B was present in the culture supernatant, the supernatant was analyzed by native PAGE and Western blotting as follows. The harvested culture supernatant was concentrated about 10-fold using a Centricon 30 concentrator (Amicon) prior to electrophoresis on native PAGE gels. The concentrated sample was then mixed with an equal volume of native gel sample buffer (50% sucrose, 0.1% bromophenol blue) and loaded on a 4-15% Tris-glycine gradient gel (Bio-Rad), along with a known sample of C. difficile toxin B, purchased from Techlab. The samples were electrophoresed for 3 hours at 150 volts, constant voltage, using a Hoefer power supply. Following electrophoresis, the gel was cut in half and one half was stained with Coomassie blue and destained with a solution comprising 10% glacial acetic acid/40% methanol to visualize any protein bands. The other half of the gel was blotted and probed using affinity purified anti-B-3 antibody (section i above).

The Coomassie blue stained gel is shown in FIG. 52. In FIG. 52, lane 1 contains broad range molecular weight markers. Lane 2 contains the commercial toxin B (Techlab); lane 3 contains BHI broth; lane 4 contains culture supernatant and lane 5 contains concentrated culture supernatant.

As shown in FIG. 52, the commercial toxin B was detectable on the Coomassie stained gel. The concentrated culture supernatant showed several relatively faint bands, however no detectable proteins in the supernatant samples co-migrated with the known C. difficile toxin B sample. Furthermore, western blot analysis did not detect any proteins in the culture supernatant that were recognized by the affinity purified anti-B-3 antibody. These results demonstrated that the above growth conditions did not appear to be optimal for toxin production.

iv) Production of C. difficile Toxin B in Dialysis Bag Culture

In order to identify growth conditions optimal for the production of C. difficile toxin B, the following experiment was conducted. A liquid culture of C. difficile #43255 was grown overnight as described above. The overnight culture was used to inoculate large scale cultures grown in dialysis bags containing PBS and submerged in BHI broth, as described by Meador and Tweten Infection and Immunity, 56:7 (1988)!. Briefly, two 500 ml wide-mouth media bottles were filled with 400 ml of BHI broth. A dialysis bag having a molecular weight cut-off of 12-14,000 (Spectrapor) containing 25 ml of PBS was tied off at both ends and submerged in the BHI media in each 500 ml bottle. The bottles and dialysis bags were then autoclaved for 30 minutes to sterilize the broth and PBS. The bottles were then incubated for 1 hour at 37° C. under anaerobic conditions to cool the liquids and remove oxygen from the media.

The PBS in each dialysis bag was then inoculated with 10 ml of the overnight culture using a 10 cc syringe fitted with a 27 gauge needle to puncture the bags above the broth level. The bottles were then incubated for 48 to 72 hours at 37° C. under anaerobic conditions. Both bottles showed heavy growth inside the dialysis bags, however one of the bottles also showed turbidity in the BHI broth outside of the dialysis bag, suggesting the BHI was contaminated. Therefore, the contents of the two dialysis bags were kept separate until the contents could be analyzed separately. It was thought that the BHI growth in the contaminated bag was due to C. difficile which may have fallen from the needle during inoculation of the dialysis bag.

v) Harvest of Dialysis Bag Culture Supernatants

The dialysis bag contents were removed and centrifuged at 5000 rpm (3440×g) for 10 minutes to pellet the cells and the dialysis bag culture supernatants from each bag were handled separately; each was filtered through a 0.45μ syringe filter and concentrated using a Centriprep 30 concentrator (Amicon). The two samples were concentrated from 35 ml to 3 ml, and stored at 2°-8° C.

The culture supernatants from the dialysis bag liquid cultures were analyzed by native PAGE and western blot analysis to evaluate the amount of toxin B produced in the dialysis bag culture supernatants.

vi) Native PAGE/Western Blot Analysis of C. difficile Dialysis Bag Culture Supernatants

Aliquots of the concentrated dialysis bag culture supernatants and a known C. difficile toxin B sample were electrophoresed on a 4-15% Tris-glycine gel (Bio-Rad) as described above in section iii). One half of the gel was stained with Coomassie blue and the other half was transferred to a nitrocellulose membrane for western blot analysis using a semi-dry transfer apparatus (Millipore)and standard transfer conditions (12 volts, constant voltage, for 30 minutes). The remaining protein binding sites on the membrane were blocked overnight in PBS containing 1 mg/ml dry milk.

FIG. 53 shows the resulting Coomassie stained gel and Western blot. In FIG. 53, lanes 1-4 represent the Coomassie stained gel and lanes 6-9 represent the Western blot. Lanes 1 and 6 contain broad range molecular weight markers; lanes 2 and 7 contain commercial toin B (Techlab); lanes 3 and 8 contain dialysis bag culture supernatant from the culture having sterile BHI broth; lanes 4 and 9 contain dialysis bag culture supernatant from the culture having "contaminated" BHI broth; lane 5 is blank.

A shown in FIG. 53, the presence of C. difficile toxin B was detected by incubating the blot strips with affinity purified anti-B-3 IgY. After washing the blots to remove unbound anti-B-3 antibodies, bound anti-B-3 antibodies were detected by incubating the strips with a secondary antibody comprising rabbit anti-chicken Ig conjugated to alkaline phosphatase (Sigma). The blots were washed again to remove any unbound secondary antibody and the blots were developed in freshly prepared BLIP/NBT substrate solution. Development was stopped by flooding the blots with water once an adequate signal was obtained.

The results of the PAGE and Western blot analysis showed that the amount of toxin B present in the dialysis bag supernatant samples was too dilute to be detected by staining with Coomassie. However, both culture supernatant samples (one from the bottle with sterile BHI broth and one from the bottle with contaminated BHI broth) contained immunoreactive toxin B when analyzed by Western blotting. The only difference seen between the two culture supernatant samples appeared to be in the amount of toxin B produced. The sterile broth sample appeared to contain more toxin B than did the contaminated broth sample.

Comparison of the commercial toxin B sample to the toxin B produced in the dialysis bag culture supernatant samples revealed that the culture supernatant sample contained a higher percentage of intact toxin B protein (i.e., there was much less evidence of degradation in the form of minor immunoreactive bands present in the culture supernatant samples). Because both culture supernatant samples contained toxin B (although at different concentrations), they were pooled prior to affinity purification.

c) Affinity Purification of C. difficile Toxin B

The dialysis bag culture supernatant samples were pooled and applied to the toxin B affinity column prepared in section a)!. Nonspecific proteins were removed by washing the column with PBS until the baseline OD was achieved. The bound protein was eluted using Actisep elution media (Sterogene) and was then dialyzed against Tris-buffered saline, pH 7.5 (50 mM Tris, 150 mM NaCl). Following dialysis, the affinity purified protein was concentrated from 40 ml to 4.5 ml using a Centricon 30 concentrator (Amicon).

d) In Vitro and In Vivo Characterization of Affinity Purified Toxin B From C. difficile

In order to determine the presence or absence of C. difficile toxin B in the Actisep-eluted sample and effluent from the affinity column (i.e., the flow-through), these samples were analyzed by native PAGE and Western blotting along with the culture supernatant starting material. These analyses were performed to evaluate the relative amount of C. difficile toxin B in the culture supernatant and the efficiency of the affinity purification.

The affinity purified, culture supernatant, flow-through, and commercial C. difficile toxin B samples were each mixed with an equal volume native sample buffer and loaded on a 4-15% native Tris-glycine gradient gel (Bio-Rad). The sample were electrophoresed for approximately 2.5 hours at 200 volts, constant voltage, using a Hoefer power supply, and transferred to nitrocellulose using a semi-dry blotting apparatus (Millipore) according to manufacturer's instructions. The blot was blocked overnight using a solution containing 1% powdered milk in PBS. The blot was then incubated with affinity purified anti-B-3 IgY as the primary antibody and rabbit anti-chicken conjugated to alkaline phosphatase as the secondary antibody. The blots were handled as described in section b(vi) to permit visualization of the C. difficile toxin B protein.

FIG. 54 shows the Coomassie stained gel and corresponding Western blots. In FIG. 54, lanes 1-3 were stained with Coomassie blue; lanes 5-10 were probed with anti-B-3 IgY and lanes 8-10 were probed with pre-immune IgY. Lanes 1, 5 and 8 contain affinity purified toxin B; lanes 2, 6 and 9 contain the column flow through; lanes 3, 7 and 10 contain commercial toxin B (Techlab). Lane 4 does not contain any protein (blank).

The following results were obtained upon western blot analysis. All three samples (culture supernatant, eluted protein and flow-through) contained immunoreactive toxin B. These results indicated that the affinity purification protocol was successful in purifying some toxin B. However, as the flow-through fraction was found to contain significant amounts of toxin B the following modifications would be examined for the ability to further optimize the purification process (e.g., coupling of B-3 IgY to Affigel hydrazide support (BioRad) via periodate oxidation of IgY).

ii) Yield of Affinity Purified C. difficile Toxin B

The yield of affinity purified C. difficile toxin B was determined by BCA protein assay (Pierce), using BSA as the protein standard. This assay showed that the toxin B concentration was 73 μg/ml×4.5 ml (volume of affinity purified material)=365 μg of toxin B. Approximately 70 ml of dialysis bag culture supernatant was used as the starting material; therefore, about 5 μg toxin B was recovered per milliliter of culture. This yield was consistent with previously reported yields using this method of culturing C. difficile 7.8 μg toxin B/ml of culture supernatant; Meador and Tweten (1988), supra!.

iii) Measurement of the In Vivo Activity of the Affinity Purified C. difficile Toxin B

The in vivo activity of the affinity purified C. difficile toxin B was determined by injecting various amounts of the purified toxin B preparation (described below) into 30 to 40 gram female syrian hamsters. Another group of hamsters was injected with various amounts of a commercial toxin B preparation (TechLabs) for comparison with results previously obtained. The LD₁₀₀ of the TechLabs preparation of C. difficile toxin B was found to be about 5 μg for 30-40 g hamsters when administered I.P. (Example 19). At this concentration (5 μg/30-40 g hamster), the hamsters died within about 3 hours post-I.P. injection.

The LD₁₀₀ concentration of the affinity purified toxin B was determined by I.P. injection of 1 ml of a solution containing either 5 or 50 μg of affinity purified toxin B diluted in saline. Two 30-40 gram hamsters were injected with each concentration of affinity purified toxin B. The hamsters injected with 50 μg of affinity purified material hamster died within 2 hours; the hamsters injected with 5 μg of affinity purified toxin B died within 4 hours. These results demonstrated that the toxicity of the affinity purified C. difficile toxin B preparation was comparable to the commercially available C. difficile toxin B.

EXAMPLE 49 Diagnostic Agglutination Assay for the Detection of C. difficile Toxin A and Toxin B

In this example, a rapid agglutination assay designed to detect C. difficile toxin A and toxin B in either culture supernatants or biological specimens such as feces was developed. Affinity purified antibodies against recombinant C. difficile toxin A and toxin B from hens were used to passively coat small polystyrene particles. In principle, the particles coated with the specific avian antibodies (IgY) to toxin A and toxin B should form visible aggregates when they are mixed with a sample containing the toxins. This format should produce a specific, sensitive and rapid assay. Affinity purified IgY in this case confers specificity and sensitivity to C. difficile toxin, while ease of use and speed of the assay is conferred using an agglutination assay format. This example describes: a) initial development of the agglutination assay for the detection of C. difficile toxin A and toxin B; and b) evaluation and optimization of the agglutination assay.

a) Development of an Agglutination Assay for Detection of C. difficile Toxin A and Toxin B

Antibodies were generated in hens using the toxin A recombinant (pMAL 1870-2680) and the toxin B recombinant (pPB1750-2360) using Freund's adjuvant as described in previous Examples. The recombinant toxin A antibodies (A-6 IgY) and the recombinant toxin B antibodies(B-3 IgY) were PEG fractionated the then affinity purified as described in Example 15c. The A-6 IgY was affinity purified against pPA1870-2680 and the B-3 IgY was affinity purified against pB1750-2369. The affinity-purified antibodies were then passively coated onto the polystyrene particles.

For each IgY preparation to be coated, 100 μl of a 5% bead suspension of 1μ beads (Spherotech Inc., Libertyville, Ill.) was removed and centrifuged for 2 minutes at 14,000×g in a Beckman microfuge to pellet the particles. The particles were then washed with TBS (10 mM Tris, 150 mM NaCl, pH 8) PBS-Tween (10 mM sodium phosphate, 150 mM NaCl, pH 7.2+0.05% Tween 20) and TBS. The particles were centrifuged for 2 minutes following each wash and the wash buffer was discarded. Following the last TBS wash, the particles were resuspended in 1 ml of the antibody coating solution; affinity purified avian A-6 or B-3 IgY at 100 μg/ml in TBS. PEG-fractionated preimmune IgY was also coated in the same manner to serve as a negative control in the agglutination assays. The particle suspensions were then inverted at room temperature for 18 to 24 hours to allow the IgY to coat the particles.

To remove the unbound antibody, the suspensions were centrifuged for 2 minutes, the antibody solution was discarded, and the particles were washed as before (TBS, PBS-Tween, TBS). After the last TBS wash, the IgY-coated particles were resuspended in 200 μl of TBS, giving a 2.5% particle suspension.

In order to demonstrate that the particles were coated with IgY, 10 μl of the particles were incubated with 5 μl of undiluted goat anti-chicken IgG (Fisher Biotech) in depression wells. Samples were then evaluated for macroscopic agglutination. Particles that had not been coated with IgY showed no agglutination in this procedure.

In order to demonstrate the feasibility of using affinity purified polyclonal avian IgY in this type of assay, the ability of A-6 IgY coated particles to agglutinate in the presence of various concentrations of toxin A was evaluated.

Commercial toxin A (Tech labs) was diluted 10-fold serially from a starting concentration of 0.29 mg/ml, using PBS containing BSA at 1 mg/ml as the diluent. Ten μl of each dilution was mixed with 10 μl of the coated beads in a depression-well slide, and the mixture was incubated for 20 minutes at 37° C. The slides were then analyzed macroscopically for evidence of agglutination.

Strong agglutination was observed with the 1:10 and 1:100 dilutions, and weak agglutination was observed in the 1:1000 dilution. The dilutions greater than 1:1000 showed no agglutination. The pre-immune coated particles did not agglutinate at any dilution tested. The 1:100 dilution of toxin A had a concentration of 2.9 μg/ml. Ten μl of the 2.9 μg/ml dilution contains 29 ng of toxin A, therefore the assay is sensitive to 29 ng of toxin A, or 2.9 μg/ml. The agglutination assay format appears to be suitable for detecting C. difficile toxins A and B.

Affinity purified polyclonal avian antibodies were most commonly used to coat the particles, however the use of PEG-fractionated and water-diluted IgY preparations was also investigated, in order to determine if it was possible to increase the sensitivity of the agglutination assay by using polyclonal antibodies which might contain a population of high affinity antibodies lost during affinity purification.

PEG-fractionated polyclonal A2 IgY was used to coat 1μ polystyrene particles under conditions identical to those described above, and the particles were evaluated for sensitivity in the C. difficile toxin A agglutination assay. These particles were less sensitive than particles coated with affinity purified IgY.

To investigate the possibility that residual PEG in the PEG-fractionated IgY may inhibit particle agglutination in the assay, A-2 IgY was extracted by the acidified water dilution method described by Akita and Nakai J. of Food Science, 57:629 (1992)!. Polystyrene particles were then coated with water diluted IgY under conditions identical to those described above, and the particles were evaluated for sensitivity in the C. difficile toxin A agglutination assay.

It was determined that particles coated with water-diluted IgY preparations were less sensitive than particles coated with affinity purified IgY. Affinity purified IgY therefore appears superior to batch-fractionated IgY preparations in this assay format. In order to increase the sensitivity and maintain the specificity of the agglutination assays, we then evaluated the effect of several other variables on the assay performance.

b) Evaluation and Optimization of the C. difficile Toxin A and Toxin B Agglutination Assay

A-6 and B-3 IgY-coated beads were evaluated for their agglutinability with lowest amount of toxin (i.e., sensitivity) and specificity. Instead of using PEG-fractionated preimmune IgY, affinity-purified IgY against an irrelevant antigen, C. atrox snake venom, was used to coat the particles as a negative control. Toxin A and toxin B were serially diluted in PBS from 1 μg/ml to 0.1 ng/ml. Ten μl of bead suspension was mixed with 20 μl sample in wells of glass agglutination plates, mixed well and rotated on nutator (Lab Quake) or manually for two minutes. Agglutination was read after two minutes. A completely uniform suspension was rated as "-," a slightly gritty appearance was rated as "±" and distinct agglutination was rated as "+" or "++," according to the size of the aggregates.

Various parameters which affect the sensitivity and/or specificity of the assay such as bead size, concentration of coating antibody, temperature of reaction, pH of coating buffer, antibodies generated using different adjuvants, final density of the beads (%, w/v) and sample diluents were evaluated. Four different bead sizes 0.39μ, 0.81μ, 1μ, and 1.2μ were initially evaluated. The 1μ bead agglutinated very rapidly, resulting in large aggregates with little or no non-specific agglutination. Hence, 1μ bead size was chosen for further optimization studies. Samples were initially diluted in PBS. If the beads autoagglutinated in PBS, PBS with 1 mg/ml BSA or PBS with 0.01% Tween-20 was substituted. Both diluents prevented autoagglutination, but PBS with Tween-20 also inhibited the specific signal. Various other blocking agents such as sucrose, BSA at higher concentration and gelatin were evaluated as diluents. PBS containing 1 mg/ml BSA was found to be optimal at preventing autoagglutination without inhibiting specific signal.

The density of the beads in final suspension was also evaluated. In order to improve the sensitivity and specificity, A-6 or B-3 IgY-coated latex particles were tested for their agglutinability at 2.5%, 1.25%, 0.5%, and 0.25% suspensions. All the bead suspensions except 2.5% resulted in no or low signal. Antibodies generated using different adjuvants have different avidities and affinities, and hence agglutinate differently. It was known that antibodies with higher avidity and affinity form large and distinct aggregates. A-6 and B-3 IgY generated using Freund's and Gerbu as adjuvants were evaluated for their agglutinability at lowest concentration of toxin. Antibodies generated using Gerbu adjuvant were found to be better in giving distinct and large aggregates at 10-times lower concentrations of toxin A or toxin B, compared to antibodies generated with Freund's adjuvant.

The effect of antibody concentration/mg of beads with lower or higher incubation temperature was also tested. The polystyrene particles were coated with 20 μg IgY or 50 μg IgY/mg of beads, and incubated at room temperature, 37° C., or 56° C. There was a direct correlation between higher concentration of coating antibody and higher temperature with respect to increase in sensitivity. However, it was found that coating the particles at higher temperature also resulted in increased non-specific signal. High pH and low pH coating buffers were evaluated in order to optimize maximum sensitivity and specificity. The polystyrene particles coated in 50 mM sodium acetate, 150 mM sodium chloride, pH 5.5 (low pH buffer) agglutinated non-specifically, while beads coated using 50 mM sodium carbonate, pH 9.5 (high pH buffer) buffer increased sensitivity and specificity with A-6 IgY coated particles, but not for B-3 IgY coated particles. The sensitivity and specificity of A-6 or B-3 IgY sensitized particles using various methods is summarized in Table 49.

                                      TABLE 49                                     __________________________________________________________________________     Summary of Results                                                                       A-6 IgY-sensitized beads      B-3 IgY-sensitized beads                         Sensitivity                   Sensitivity                                        100 50  10  1   neg Specificity                                                                            100                                                                               10  1   neg Specificity             Conditions  ng/ml                                                                              ng/ml                                                                              ng/ml                                                                              ng/ml                                                                              control                                                                            Feces.sup.M                                                                         E. coli                                                                           ng/ml                                                                             ng/ml                                                                              ng/ml                                                                              control                                                                            Feces.sup.M                                                                         E.                 __________________________________________________________________________                                                                coli                A. 100 μg/ml, 1μ, TBS,                                                               ++  -   -   -   -   -   -   ++ +   -   -   -   -                   pH 7.5 (F/RT)                                                                  B. 100 μg/ml, 1μ, TBS,                                                               ++  ±                                                                               -   -   -   ++  ++  ++ ++  ±                                                                               -   -   -                   pH 7.5 (G/RT)                                                                  C. 100 μg/ml, 1μ, TBS,                                                               ++  ++  +   -   -   ++  ++  n/a                                    pH 7.5 (G/37° C.)                                                       D. 250 μg/ml, 1μ, TBS,                                                               ++  +   -   -   -   ++  ++  n/a                                    pH 7.5 (G/RT)                                                                  E. 250 μg/ml, 1μ, TBS,                                                               ++  ++  ++  -   -   ++  ++  ++ ++  ++  +*  ++  ++                  pH 7.5, (G/37° C.)                                                      F. 30 μg/mg of beads                                                                    ±                                                                               -   -   -   -   ++  ++  ±                                                                              -   -   -   -   ∓                0.81μ (Yamamoto,                                                            et al procedure                                                                G. 30 μg/mg of beads                                                                    ±                                                                               -   -   -   -   ++  ++  ±                                                                              -   -   -   -   ∓                0.39μ (Yamamoto,                                                            et al procedure)                                                               H. 150 μg/ml, 1μ, TBS,                                                               ++  ++  +   -   -   ++  ++  +  ∓                                                                               ∓                                                                               ∓                                                                               ++  ++                  pH 7.5 (G/RT)                                                                  blocking procedure                                                             I. 150 μg/ml, 0.81μ,                                                                 ++  ±                                                                               -   -   -   ++  +   +  ±                                                                               ∓                                                                               ∓                                                                               ++  +                   TBS, pH 7.5, (G/RT)                                                            blocking procedure                                                             J. 100 μg/ml, 1μ, TBS,                                                               ++  +   -   -   -   -   -   n/a                                    pH 7.5, (F/RT)                                                                 K. 100 μg/ml, 1μ, TBS,                                                               ++  ++  ±                                                                               -   -   ++  ++  n/a                                    pH 7.5 (G/RT)                                                                  L. 250 μg/ml, 1μ, TBS,                                                               ++  ++  ±                                                                               -   -   ±                                                                               ±                                                                               n/a                                    pH 7.5 (F/RT)                                                                  M. 250 μg/ml, 1μ,                                                                    ++  ++  ±                                                                               -   -   ++  ++  n/a                                    Acetate, pH 5.5                                                                (F/RT)                                                                         N. 250 μg/ml, 1μ, CO.sub.3                                                           ++  ++  -   -   -   -   -                                          buffer, pH 9.5, (F/RT)                                                         O. 250 μg/ml, 1μ, CO.sub.3                                                           ++  ++  +   -   -   -   -   ++ ±                                                                               -   -   -   -                   buffer, pH 9.5,                                                                (G/RT)                                                                         P. 250 μg/ml, 1μ,                                                                    ++  ++  ++  -   +   ++  ++  n/a                                    Glycine saline buffer,                                                         pH 8.2 (F/56°C.)                                                        Q. 250 μg/ml, 1.2μ, pH                                                               ++  ++  ++  +   -   ++  +   ++ ++  +   +   ++  +                   9.5, (F/RT)                                                                    R. same as Q except                                                                        ++  ++  ++  -   -   ++  +   ++ ++  +   +   +   +                   after overcoating                                                              w/BSA                                                                          __________________________________________________________________________      Explaination of conditions: coating concentration of IgY, bead size,           coating solution, pH, adjuvant used, temperature during coating.               *No autoagglutination with sensitivity of 1 ng/ml in presence of PBS + BS      + 0.01% TW20, however nonspecific agglutination did occur with E. coli an      M feces even with this diluent.                                                F = IgY generated using Freund's adjuvant                                      G = IgY generated using Gerbu adjuvant                                         E. coli = E. coli colony was picked from agar plate and resuspended in 50      μl diluent                                                                  Feces.sup.M = 50 mg mouse feces was suspended in 500 μl diluent             vortexed and spun at 14,000 × g for 2 min. supernatant was used in       the assay                                                                

Based on the information from various parameters tested, the following bead coating protocols with A-6 and B-3 IgY were established:

i) A-6 IgY coated particles to detect toxin A directly from feces of human patients.

Five mg polystyrene particles (1μ, Spherotech Inc., Libertyville, Ill.) were added to a tube and washed with 1 ml of TBS, PBS-T, and TBS followed by another wash with 50 mM Na₂ CO₃, pH 9.5 buffer. The beads were resuspended in the latter buffer to a total volume of 1 ml. A-6 IgY (affinity-purified, Gerbu-generated) was added to beads to a final concentration of 250 μg/ml and incubated at room temperature on a nutator overnight. The next day, IgY-sensitized particles were washed with TBS, PBS-T, and TBS and resuspended in TBS to a final concentration of 2.5%. These IgY-sensitized particles were stored at 4° C. until use.

ii) B-3 coated latex particles to detect toxin B directly from feces of human patients.

Five mg polystyrene particles (1μ, Spherotech Inc., Libertyville, Ill.) were added to a tube and washed with 1 ml of TBS, PBS-T, and TBS followed by another wash with 50 mM Na₂ CO₃, pH 9.5 buffer. The beads were resuspended in the latter buffer to a total volume of 1 ml. B-3 IgY (affinity-purified, Gerbu-generated) was added to beads to a final concentration of 100 μg/ml and incubated at room temperature on a nutator overnight. The next day, IgY-sensitized latex particles were washed with TBS, PBS-T, and TBS and resuspended in TBS to a final concentration of 2.5%. These IgY-sensitized latex particles were stored at 4° C. until use.

The agglutination assay to detect toxin A and B from feces was compared with commercially available assays to detect toxin A and toxin B from human stool specimens. Cyctoclone™ A+B EIA (Cambridge Biotech), which detects both toxins A and B, and Premier™ C. difficile Toxin A Test (Meridian Diagnostics Inc.), which detects only toxin A, were used for comparison. Normal human stool specimens were processed according to each of the manufacturer's instructions. Stool samples were spiked with 1 μg/ml of toxin A or toxin B and serially diluted 10-fold, to 0.01 ng/ml toxin A and 0.1 ng/ml toxin B. For agglutination assays, stool was diluted 5-fold with PBS containing 1 mg/ml BSA and centrifuged at 2500 ×g for 3 minutes. The supernatant was then used in the assay.

EIA's were performed according to the manufacturer's instructions, and results were read spectrophotometrically. Interpretation of results was made based on optical density values and the manufacturer's recommendations. For the agglutination assay, 10 μl suspension of A-6, B-3 IgY- or non-specific IgY-sensitized particles were placed in the wells of glass agglutination plates. Twenty μl samples were added to each well, mixed well, and rotated on a nutator. Agglutination was read visually after 2 minutes of rotation. Interpretation of results was made as described earlier. The summary of the results is presented in Table 50. The agglutination assay of the present invention detected toxin A at 1 ng/ml, while both the Cytoclone™ A+B EIA, and Premier™ C. difficile toxin A test detected toxins at 10-fold lower levels. Toxin B was detected at 1 ng/ml, using both agglutination and Cytoclone A+B EIA. The results show that agglutination assay of the invention, is simple, easy to perform, and very rapid, as the results can be obtained in 5 minutes.

                                      TABLE 50                                     __________________________________________________________________________     Comparison of results of three different methods to detect                     toxin A and toxin B spiked in normal human stool specimens                                                   Agglutination assay for                                   Cytoclone ™                                                                           Premier ™ C. difficle                                                                  Toxin A & B                                               A + B EIA toxin A test                                                                              Ophidian                                         Parameter                                                                               Cambridge Biotech                                                                        Meridian Diagnostics                                                                      Pharmaceuticals                                  __________________________________________________________________________     Stool spiked with                                                              Toxin A                                                                        100 ng/ml                                                                               ++        ++         ++                                               10 ng/ml ++        ++         ++                                               1 ng/ml  ++        ++         +                                                0.1 ng/ml                                                                               +         ++         -                                                0.01 ng/ml                                                                              -         ND         ND                                               Stool spiked with                                                              Toxin B                                                                        100 ng/ml                                                                               ++        N/A        ++                                               10 ng/ml ++                   ++                                               1 ng/ml  ±                 ±                                             0.1 ng/ml                                                                               -                    -                                                Total Time                                                                              150 min   150 min    5 min                                            __________________________________________________________________________      ND Not determined                                                              N/A Not applicable                                                       

EXAMPLE 50 Characterization of Hamsters After Successful Treatment with Avian Antibodies Directed against Recombinant C. difficile Toxin A and Toxin B Proteins

In order to investigate why hamsters treated with IgYs directed against recombinant toxin A (A-6 IgY) and recombinant toxin B (B-3 IgY) before or after challenge with C. difficile do not relapse and contract C. difficile associated disease CDAD) after the withdrawal of treatment, the following experiment was performed.

Relapse is commonly seen in hamsters (as demonstrated in Example 33) and in humans treated with drugs, such as vancomycin or metronidazole, to combat CDAD once drug treatment is terminated. In contrast, data presented in Examples 16 and 32 show that IgYs directed against recombinant C. difficile toxin A alone (given prophylactically) or a mixture containing IgYs directed against recombinant toxin A and B proteins (given therapeutically) can be used to successfully prevent or treat CDAD and also prevent relapse.

The example involved a) the detection of C. difficile organisms and toxins in the feces of hamsters treated with anti-A-6/B-3 IgY, b) the detection of anti-C. difficile toxin A and anti-C. difficile toxin B IgG in the serum of treated hamsters by ELISA, c) the detection of anti-C. difficile toxin A and anti-C. difficile toxin B IgA in the saliva of treated hamsters by ELISA and d) re-exposure of A-6/B-3 treated hamsters with antibiotics.

a) Detection of C. difficile Organisms and Toxins in the Feces of Hamsters Treated with A-6/B-3 IgY

The 7 hamsters that were successfully treated with 2 ml per day of A-6/B-3 IgY and the lone surviving hamster treated with 1 ml per day of A-6/B-3 IgY (Example 42) were tested for the presence of C. difficile and toxin A and toxin B in fecal material after treatment was withdrawn. This determination was performed to investigate whether hamsters treated A-6/B-3 IgY were protected from relapse because the IgY treatment either reduced or completely eliminated C. difficile organisms and toxins from the GI tract of the treated hamsters.

Stools were collected from the 7 individual hamsters 4 days after termination of treatment with A-6/B-3 IgY. A suspension was made from the stool samples as follows. Fifty milligrams of feces were added to 100 μl of PBS (pH 7.4) and the mixture was suspended by vortexing the sample. An aliquot (50 μl) of each suspension was streaked unto a C. difficile selective agar plate (CCFA plates;BBL) and the plates were incubated for 48 hours under anaerobic conditions. The remaining suspension was tested for the presence of C. difficile toxin A and toxin B using the toxin agglutination assay described in Example 49.

The results obtained by culturing stool suspensions on the CCFA plates demonstrated that all of the hamsters successfully treated with A-6/B-3 IgY still harbored C. difficile organisms 4 days after treatment (ranging from approximately 6-100 colonies). Furthermore, C. difficile toxin A was detected in the feces from all nine treated hamsters using the agglutination assay (Example 49). Surprisingly, C. difficile toxin B was not detected in the feces of any of the hamsters.

Stool samples were collected from the same 7 hamsters again about 5 weeks after the termination of antibody treatment. Suspensions were prepared and plated onto CCFA plates as described above. After this prolonged period, the presence of C. difficile was only detected in the stool from one of the hamsters. Interestingly, the organisms were detected in the hamster that was treated with the lower (1 ml) dose of A-6/B-3 IgY. Only a low number of colonies (5 colonies) was detected in the stool of that animal. In control animals there were no organisms detected, as normally, only a very low percentage of hamsters have detectable levels of organisms.

These results indicate that although the A-6/B-3 treated hamsters have been successfully treated for CDAD and the disease does not relapse, they still shed C. difficile organisms and contain toxin A in their feces early after treatment. The anti-recombinant C. difficile toxin A and B antibodies (i.e., A-6/B-3 IgY) apparently eliminate disease symptoms without completely eliminating C. difficile organisms or toxin A from the GI tract of the treated hamsters. While not limiting the invention to a particular theory of action, the avian IgYs may exert their therapeutic effects by lowering the level of toxin present and thus possibly reducing organism number enough to not only prevent CDAD but also prevent CDAD from re-occurring, as it is possible that toxin A may aid in the colonization of C. difficile.

Five weeks after treatment with the avian antitoxin preparation, C. difficile organisms were not detected in the feces of most (7/8) of the treated hamsters. These results indicate that long-term colonization of the GI tract by C. difficile does not occur following treatment with A-6/B-3 IgY.

b) Detection of Anti-C. difficile Toxin A and Anti-C. difficile Toxin B IgG in the Serum of Treated Hamsters by ELISA

Serum was collected from hamsters following treatment with anti-A-6/B-3 IgY to determine if an endogenous serum IgG response directed against C. difficile toxins was elicited in the treated hamsters. The generation of an anti-toxin IgG response could account for the prevention of subsequent relapse in the animals.

Blood was collected by cardiac puncture in the seven hamsters that were still available after five weeks (described above) four days after termination of treatment. The blood was allowed to clot and serum was prepared by centrifugation of the clotted sample. Serum was also collected from an uninfected hamster and from a hamster vaccinated with a mixture of recombinant toxin A and toxin B proteins (Example 39) to serve as negative and positive controls, respectively. The ELISA was conducted using the protocol described in Example 1. Briefly, the wells of the microtiter plates were coated with 0.05 μg/ml of the toxin A recombinant pPA1870-2680 (A-6) or 1.0 μg/ml of toxin B recombinant pPB 1750-2360 (B-3) at 100 μl per well. Serum samples were tested at a starting dilution of 1:50 followed by 5-fold serial dilutions. Goat-anti-hamster IgG alkaline phosphatase (Southern Biotechnology Assoc.) was used at a dilution of 1:1000 as the secondary antibody. All antibody incubations were carried out at 37° C. for 2 hours. The plates were developed for 30 minutes using para-nitrophenyl phosphate (Sigma).

The results of the ELISA demonstrated that all of the serum samples from the test hamsters contained significantly lower levels of anti-toxin A and anti-toxin B IgG as compared to the positive control serum (serum from a hamster that generated a protective IgG response after active immunization with recombinant toxin A and B proteins). Antibody titers present in the serum from the 7 treated hamsters were comparable to those present in the negative control. These results demonstrated that the protection from CDAD relapse achieved by treatment of hamsters with the A-6/B-3 IgY is probably not due to the generation of an active serum IgG response in the hamsters following infection with C. difficile organisms.

c) Detection of an Anti-C. difficile Toxin A and Anti-C. difficile Toxin B IgA Response in the Saliva by ELISA in Treated Hamsters

To investigate whether the protection from relapse from CDAD seen in the anti-A-6/B-3 IgY treated hamsters was due to the production of a mucosal IgA response in the animals, the following experiment was performed. Saliva was collected from 6 hamsters previously treatment with anti-A-6/B-3 IgY (Example 42; 2 ml) using pilocarpine (Sigma) which causes hyper-secretion of saliva. Hamsters were injected I.P. with a solution containing pilocarpine (1 mg/ml) in sterile water; 1 to 3 mgs pilocarpine was administered to the animals. Saliva was collected from 6 hamsters using a pipettor. As a negative control, saliva was collected from an mouse given 200 μg of pilocarpine. A mouse specimen was used as a negative control because the only anti-IgA conjugate commercially available is a goat anti-mouse IgA (this reagent has been reported to cross-react with hamster IgA).

The ELISA was performed as described above (b) with the following modifications. The saliva samples were tested at an initial 1:10 dilution followed by serial five-fold dilutions. Goat anti-mouse IgA (Southern Biotechnology Assoc.) was used as the secondary antibody at a 1:1000 dilution.

The results of the ELISA showed that saliva from only 2 out of the 6 treated hamsters contained levels of anti-toxin A and anti-toxin B IgA higher than that seen in the mouse negative control. The saliva of the two anti-toxin IgA-positive hamsters had fairly low titers (between 1:250 and 1:1250). Typical hyper-immune IgA titers normally range about 1:10,000 or greater. The remaining 4 hamsters did not have a significant anti-toxin IgA response to either toxin A or toxin B as compared to the negative control. Since all six of the hamsters were successfully treated against CDAD relapse and the majority (4/6) of the treated hamsters did not produce a significant anti-toxin IgA response, it is unlikely that the prevention of relapse was due to the generation of an anti-toxin A or anti-toxin B IgA response by the hamster.

The results shown in sections b) and c) indicate that the protection against relapse seen in hamsters successfully treated with IgY directed against C. difficile Intervals A-6 and B-3 is not due to the production of a anti-C. difficile toxin humoral response by the host. Thus, prevention of relapse is a function of the administration of the IgY preparations. This indicates that the host's immune status may not be relevant in terms of prediction of disease outcome (i.e., survival) or whether relapse will occur. This is important as many of the patients who would most benefit most from treatment with an A-6/B-3 IgY therapeutic are immunocompromised.

d) Re-exposure of A-6/B-3 Treated Hamsters with Antibiotics

As shown in section a) above, the treated hamsters still possess detectable levels of C. difficile organisms in their feces (4 days after termination of IgY treatment) and thus have the potential for developing CDAD. An experiment was performed to investigate whether re-exposure of these treated hamsters to Clindamycin would initiate onset of CDAD.

Four of the same hamsters used in the above experiments (e.g., in Example 42) that were successfully treated with A-6/B-3 IgY were again predisposed to C. difficile infection by administration of Clindamycin-phosphate. Seven days after termination of the initial antibody treatment, the hamsters were given another I.P. injection of Clindamycin-phosphate (Biomol) at 1 mg/100 g body weight. Twelve days post-Clindamycin predisposition (i.e., the second application of Clindamycin), none of the hamsters developed any signs of CDAD.

These results demonstrated that once the hamsters are successfully treated with the anti-A-6/B-3 IgY, they were resistant to developing CDAD even after another exposure to Clindamycin. To take this result another step further, the same hamsters were given another antibiotic, namely Cefoxitin (Sigma) which is also known to predispose the hamsters to C. difficile infection. This was done as it was possible that the prevention of CDAD in the hamsters after the Clindamycin re-treatment was due to the generation of Clindamycin-resistant normal flora which may have prevented colonization of the GI tract with C. difficile. The 4 hamsters were each given a subcutaneous injection of 10 mg of Cefoxitin in saline 11 days after (18 days post treatment with A-6/B-3 IgY). This dosage of Cefoxitin is known to predispose hamsters to CDAD.

Seven days post-Cefoxitin treatment, 1 of the 4 hamsters developed diarrhea and died. The remaining 3 hamsters remained healthy and have survived long-term (i.e., at least one month). The results obtained after treatment with Cefoxitin indicate that protection from the re-occurrence of CDAD in the treated hamsters is probably not due to the development of specific antibiotic resistance (i.e., resistance to Clindamycin) in the hamster flora.

Together the above results showed that hamsters treated for CDAD using anti-A-6/B-3 IgY contain viable C. difficile organisms and C. difficile toxin A in their GI tract early after the withdrawal of treatment and yet the hamsters do not relapse. Even more surprising was the finding that while the hamsters still harbor C. difficile in the gut, they were resistant to a subsequent challenge using antibiotics capable of predisposing hamsters to CDAD. As was shown above, 5 weeks after withdrawal of the avian antitoxin, the hamsters no longer shed organisms into feces and thus were probably no longer colonized by C. difficile. The results further indicate that oral administration of A-6/B-3 IgY to hamsters not only successfully treated CDAD, but also conferred resistance to relapse. Moreover, the A-6/B-3 IgY protected the hamsters against CDAD from repeated antibiotic predisposition using two different antibiotics.

From the above it is clear that the present invention provides antitoxins and vaccines for the treatment and prevention of C. difficile disease. Furthermore, these antitoxins prevent the relapse of C. difficile disease which is commonly seen using conventional treatment protocols. Additionally, the invention provides a rapid agglutination assay for the detection of C. difficile toxins A and B in samples.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 32                                                  (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 24 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        GGAAATTTAGCTGCAGCATCTGAC24                                                     (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 24 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        TCTAGCAAATTCGCTTGTGTTGAA24                                                     (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        CTCGCATATAGCATTAGACC20                                                         (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 19 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        CTATCTAGGCCTAAAGTAT19                                                          (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 8133 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..8130                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        ATGTCTTTAATATCTAAAGAAGAGTTAATAAAACTCGCATATAGCATT48                             MetSerLeuIleSerLysGluGluLeuIleLysLeuAlaTyrSerIle                               151015                                                                         AGACCAAGAGAAAATGAGTATAAAACTATACTAACTAATTTAGACGAA96                             ArgProArgGluAsnGluTyrLysThrIleLeuThrAsnLeuAspGlu                               202530                                                                         TATAATAAGTTAACTACAAACAATAATGAAAATAAATATTTGCAATTA144                            TyrAsnLysLeuThrThrAsnAsnAsnGluAsnLysTyrLeuGlnLeu                               354045                                                                         AAAAAACTAAATGAATCAATTGATGTTTTTATGAATAAATATAAAACT192                            LysLysLeuAsnGluSerIleAspValPheMetAsnLysTyrLysThr                               505560                                                                         TCAAGCAGAAATAGAGCACTCTCTAATCTAAAAAAAGATATATTAAAA240                            SerSerArgAsnArgAlaLeuSerAsnLeuLysLysAspIleLeuLys                               65707580                                                                       GAAGTAATTCTTATTAAAAATTCCAATACAAGCCCTGTAGAAAAAAAT288                            GluValIleLeuIleLysAsnSerAsnThrSerProValGluLysAsn                               859095                                                                         TTACATTTTGTATGGATAGGTGGAGAAGTCAGTGATATTGCTCTTGAA336                            LeuHisPheValTrpIleGlyGlyGluValSerAspIleAlaLeuGlu                               100105110                                                                      TACATAAAACAATGGGCTGATATTAATGCAGAATATAATATTAAACTG384                            TyrIleLysGlnTrpAlaAspIleAsnAlaGluTyrAsnIleLysLeu                               115120125                                                                      TGGTATGATAGTGAAGCATTCTTAGTAAATACACTAAAAAAGGCTATA432                            TrpTyrAspSerGluAlaPheLeuValAsnThrLeuLysLysAlaIle                               130135140                                                                      GTTGAATCTTCTACCACTGAAGCATTACAGCTACTAGAGGAAGAGATT480                            ValGluSerSerThrThrGluAlaLeuGlnLeuLeuGluGluGluIle                               145150155160                                                                   CAAAATCCTCAATTTGATAATATGAAATTTTACAAAAAAAGGATGGAA528                            GlnAsnProGlnPheAspAsnMetLysPheTyrLysLysArgMetGlu                               165170175                                                                      TTTATATATGATAGACAAAAAAGGTTTATAAATTATTATAAATCTCAA576                            PheIleTyrAspArgGlnLysArgPheIleAsnTyrTyrLysSerGln                               180185190                                                                      ATCAATAAACCTACAGTACCTACAATAGATGATATTATAAAGTCTCAT624                            IleAsnLysProThrValProThrIleAspAspIleIleLysSerHis                               195200205                                                                      CTAGTATCTGAATATAATAGAGATGAAACTGTATTAGAATCATATAGA672                            LeuValSerGluTyrAsnArgAspGluThrValLeuGluSerTyrArg                               210215220                                                                      ACAAATTCTTTGAGAAAAATAAATAGTAATCATGGGATAGATATCAGG720                            ThrAsnSerLeuArgLysIleAsnSerAsnHisGlyIleAspIleArg                               225230235240                                                                   GCTAATAGTTTGTTTACAGAACAAGAGTTATTAAATATTTATAGTCAG768                            AlaAsnSerLeuPheThrGluGlnGluLeuLeuAsnIleTyrSerGln                               245250255                                                                      GAGTTGTTAAATCGTGGAAATTTAGCTGCAGCATCTGACATAGTAAGA816                            GluLeuLeuAsnArgGlyAsnLeuAlaAlaAlaSerAspIleValArg                               260265270                                                                      TTATTAGCCCTAAAAAATTTTGGCGGAGTATATTTAGATGTTGATATG864                            LeuLeuAlaLeuLysAsnPheGlyGlyValTyrLeuAspValAspMet                               275280285                                                                      CTTCCAGGTATTCACTCTGATTTATTTAAAACAATATCTAGACCTAGC912                            LeuProGlyIleHisSerAspLeuPheLysThrIleSerArgProSer                               290295300                                                                      TCTATTGGACTAGACCGTTGGGAAATGATAAAATTAGAGGCTATTATG960                            SerIleGlyLeuAspArgTrpGluMetIleLysLeuGluAlaIleMet                               305310315320                                                                   AAGTATAAAAAATATATAAATAATTATACATCAGAAAACTTTGATAAA1008                           LysTyrLysLysTyrIleAsnAsnTyrThrSerGluAsnPheAspLys                               325330335                                                                      CTTGATCAACAATTAAAAGATAATTTTAAACTCATTATAGAAAGTAAA1056                           LeuAspGlnGlnLeuLysAspAsnPheLysLeuIleIleGluSerLys                               340345350                                                                      AGTGAAAAATCTGAGATATTTTCTAAATTAGAAAATTTAAATGTATCT1104                           SerGluLysSerGluIlePheSerLysLeuGluAsnLeuAsnValSer                               355360365                                                                      GATCTTGAAATTAAAATAGCTTTCGCTTTAGGCAGTGTTATAAATCAA1152                           AspLeuGluIleLysIleAlaPheAlaLeuGlySerValIleAsnGln                               370375380                                                                      GCCTTGATATCAAAACAAGGTTCATATCTTACTAACCTAGTAATAGAA1200                           AlaLeuIleSerLysGlnGlySerTyrLeuThrAsnLeuValIleGlu                               385390395400                                                                   CAAGTAAAAAATAGATATCAATTTTTAAACCAACACCTTAACCCAGCC1248                           GlnValLysAsnArgTyrGlnPheLeuAsnGlnHisLeuAsnProAla                               405410415                                                                      ATAGAGTCTGATAATAACTTCACAGATACTACTAAAATTTTTCATGAT1296                           IleGluSerAspAsnAsnPheThrAspThrThrLysIlePheHisAsp                               420425430                                                                      TCATTATTTAATTCAGCTACCGCAGAAAACTCTATGTTTTTAACAAAA1344                           SerLeuPheAsnSerAlaThrAlaGluAsnSerMetPheLeuThrLys                               435440445                                                                      ATAGCACCATACTTACAAGTAGGTTTTATGCCAGAAGCTCGCTCCACA1392                           IleAlaProTyrLeuGlnValGlyPheMetProGluAlaArgSerThr                               450455460                                                                      ATAAGTTTAAGTGGTCCAGGAGCTTATGCGTCAGCTTACTATGATTTC1440                           IleSerLeuSerGlyProGlyAlaTyrAlaSerAlaTyrTyrAspPhe                               465470475480                                                                   ATAAATTTACAAGAAAATACTATAGAAAAAACTTTAAAAGCATCAGAT1488                           IleAsnLeuGlnGluAsnThrIleGluLysThrLeuLysAlaSerAsp                               485490495                                                                      TTAATAGAATTTAAATTCCCAGAAAATAATCTATCTCAATTGACAGAA1536                           LeuIleGluPheLysPheProGluAsnAsnLeuSerGlnLeuThrGlu                               500505510                                                                      CAAGAAATAAATAGTCTATGGAGCTTTGATCAAGCAAGTGCAAAATAT1584                           GlnGluIleAsnSerLeuTrpSerPheAspGlnAlaSerAlaLysTyr                               515520525                                                                      CAATTTGAGAAATATGTAAGAGATTATACTGGTGGATCTCTTTCTGAA1632                           GlnPheGluLysTyrValArgAspTyrThrGlyGlySerLeuSerGlu                               530535540                                                                      GACAATGGGGTAGACTTTAATAAAAATACTGCCCTCGACAAAAACTAT1680                           AspAsnGlyValAspPheAsnLysAsnThrAlaLeuAspLysAsnTyr                               545550555560                                                                   TTATTAAATAATAAAATTCCATCAAACAATGTAGAAGAAGCTGGAAGT1728                           LeuLeuAsnAsnLysIleProSerAsnAsnValGluGluAlaGlySer                               565570575                                                                      AAAAATTATGTTCATTATATCATACAGTTACAAGGAGATGATATAAGT1776                           LysAsnTyrValHisTyrIleIleGlnLeuGlnGlyAspAspIleSer                               580585590                                                                      TATGAAGCAACATGCAATTTATTTTCTAAAAATCCTAAAAATAGTATT1824                           TyrGluAlaThrCysAsnLeuPheSerLysAsnProLysAsnSerIle                               595600605                                                                      ATTATACAACGAAATATGAATGAAAGTGCAAAAAGCTACTTTTTAAGT1872                           IleIleGlnArgAsnMetAsnGluSerAlaLysSerTyrPheLeuSer                               610615620                                                                      GATGATGGAGAATCTATTTTAGAATTAAATAAATATAGGATACCTGAA1920                           AspAspGlyGluSerIleLeuGluLeuAsnLysTyrArgIleProGlu                               625630635640                                                                   AGATTAAAAAATAAGGAAAAAGTAAAAGTAACCTTTATTGGACATGGT1968                           ArgLeuLysAsnLysGluLysValLysValThrPheIleGlyHisGly                               645650655                                                                      AAAGATGAATTCAACACAAGCGAATTTGCTAGATTAAGTGTAGATTCA2016                           LysAspGluPheAsnThrSerGluPheAlaArgLeuSerValAspSer                               660665670                                                                      CTTTCCAATGAGATAAGTTCATTTTTAGATACCATAAAATTAGATATA2064                           LeuSerAsnGluIleSerSerPheLeuAspThrIleLysLeuAspIle                               675680685                                                                      TCACCTAAAAATGTAGAAGTAAACTTACTTGGATGTAATATGTTTAGT2112                           SerProLysAsnValGluValAsnLeuLeuGlyCysAsnMetPheSer                               690695700                                                                      TATGATTTTAATGTTGAAGAAACTTATCCTGGGAAGTTGCTATTAAGT2160                           TyrAspPheAsnValGluGluThrTyrProGlyLysLeuLeuLeuSer                               705710715720                                                                   ATTATGGACAAAATTACTTCCACTTTACCTGATGTAAATAAAAATTCT2208                           IleMetAspLysIleThrSerThrLeuProAspValAsnLysAsnSer                               725730735                                                                      ATTACTATAGGAGCAAATCAATATGAAGTAAGAATTAATAGTGAGGGA2256                           IleThrIleGlyAlaAsnGlnTyrGluValArgIleAsnSerGluGly                               740745750                                                                      AGAAAAGAACTTCTGGCTCACTCAGGTAAATGGATAAATAAAGAAGAA2304                           ArgLysGluLeuLeuAlaHisSerGlyLysTrpIleAsnLysGluGlu                               755760765                                                                      GCTATTATGAGCGATTTATCTAGTAAAGAATACATTTTTTTTGATTCT2352                           AlaIleMetSerAspLeuSerSerLysGluTyrIlePhePheAspSer                               770775780                                                                      ATAGATAATAAGCTAAAAGCAAAGTCCAAGAATATTCCAGGATTAGCA2400                           IleAspAsnLysLeuLysAlaLysSerLysAsnIleProGlyLeuAla                               785790795800                                                                   TCAATATCAGAAGATATAAAAACATTATTACTTGATGCAAGTGTTAGT2448                           SerIleSerGluAspIleLysThrLeuLeuLeuAspAlaSerValSer                               805810815                                                                      CCTGATACAAAATTTATTTTAAATAATCTTAAGCTTAATATTGAATCT2496                           ProAspThrLysPheIleLeuAsnAsnLeuLysLeuAsnIleGluSer                               820825830                                                                      TCTATTGGGGATTACATTTATTATGAAAAATTAGAGCCTGTTAAAAAT2544                           SerIleGlyAspTyrIleTyrTyrGluLysLeuGluProValLysAsn                               835840845                                                                      ATAATTCACAATTCTATAGATGATTTAATAGATGAGTTCAATCTACTT2592                           IleIleHisAsnSerIleAspAspLeuIleAspGluPheAsnLeuLeu                               850855860                                                                      GAAAATGTATCTGATGAATTATATGAATTAAAAAAATTAAATAATCTA2640                           GluAsnValSerAspGluLeuTyrGluLeuLysLysLeuAsnAsnLeu                               865870875880                                                                   GATGAGAAGTATTTAATATCTTTTGAAGATATCTCAAAAAATAATTCA2688                           AspGluLysTyrLeuIleSerPheGluAspIleSerLysAsnAsnSer                               885890895                                                                      ACTTACTCTGTAAGATTTATTAACAAAAGTAATGGTGAGTCAGTTTAT2736                           ThrTyrSerValArgPheIleAsnLysSerAsnGlyGluSerValTyr                               900905910                                                                      GTAGAAACAGAAAAAGAAATTTTTTCAAAATATAGCGAACATATTACA2784                           ValGluThrGluLysGluIlePheSerLysTyrSerGluHisIleThr                               915920925                                                                      AAAGAAATAAGTACTATAAAGAATAGTATAATTACAGATGTTAATGGT2832                           LysGluIleSerThrIleLysAsnSerIleIleThrAspValAsnGly                               930935940                                                                      AATTTATTGGATAATATACAGTTAGATCATACTTCTCAAGTTAATACA2880                           AsnLeuLeuAspAsnIleGlnLeuAspHisThrSerGlnValAsnThr                               945950955960                                                                   TTAAACGCAGCATTCTTTATTCAATCATTAATAGATTATAGTAGCAAT2928                           LeuAsnAlaAlaPhePheIleGlnSerLeuIleAspTyrSerSerAsn                               965970975                                                                      AAAGATGTACTGAATGATTTAAGTACCTCAGTTAAGGTTCAACTTTAT2976                           LysAspValLeuAsnAspLeuSerThrSerValLysValGlnLeuTyr                               980985990                                                                      GCTCAACTATTTAGTACAGGTTTAAATACTATATATGACTCTATCCAA3024                           AlaGlnLeuPheSerThrGlyLeuAsnThrIleTyrAspSerIleGln                               99510001005                                                                    TTAGTAAATTTAATATCAAATGCAGTAAATGATACTATAAATGTACTA3072                           LeuValAsnLeuIleSerAsnAlaValAsnAspThrIleAsnValLeu                               101010151020                                                                   CCTACAATAACAGAGGGGATACCTATTGTATCTACTATATTAGACGGA3120                           ProThrIleThrGluGlyIleProIleValSerThrIleLeuAspGly                               1025103010351040                                                               ATAAACTTAGGTGCAGCAATTAAGGAATTACTAGACGAACATGACCCA3168                           IleAsnLeuGlyAlaAlaIleLysGluLeuLeuAspGluHisAspPro                               104510501055                                                                   TTACTAAAAAAAGAATTAGAAGCTAAGGTGGGTGTTTTAGCAATAAAT3216                           LeuLeuLysLysGluLeuGluAlaLysValGlyValLeuAlaIleAsn                               106010651070                                                                   ATGTCATTATCTATAGCTGCAACTGTAGCTTCAATTGTTGGAATAGGT3264                           MetSerLeuSerIleAlaAlaThrValAlaSerIleValGlyIleGly                               107510801085                                                                   GCTGAAGTTACTATTTTCTTATTACCTATAGCTGGTATATCTGCAGGA3312                           AlaGluValThrIlePheLeuLeuProIleAlaGlyIleSerAlaGly                               109010951100                                                                   ATACCTTCATTAGTTAATAATGAATTAATATTGCATGATAAGGCAACT3360                           IleProSerLeuValAsnAsnGluLeuIleLeuHisAspLysAlaThr                               1105111011151120                                                               TCAGTGGTAAACTATTTTAATCATTTGTCTGAATCTAAAAAATATGGC3408                           SerValValAsnTyrPheAsnHisLeuSerGluSerLysLysTyrGly                               112511301135                                                                   CCTCTTAAAACAGAAGATGATAAAATTTTAGTTCCTATTGATGATTTA3456                           ProLeuLysThrGluAspAspLysIleLeuValProIleAspAspLeu                               114011451150                                                                   GTAATATCAGAAATAGATTTTAATAATAATTCGATAAAACTAGGAACA3504                           ValIleSerGluIleAspPheAsnAsnAsnSerIleLysLeuGlyThr                               115511601165                                                                   TGTAATATATTAGCAATGGAGGGGGGATCAGGACACACAGTGACTGGT3552                           CysAsnIleLeuAlaMetGluGlyGlySerGlyHisThrValThrGly                               117011751180                                                                   AATATAGATCACTTTTTCTCATCTCCATCTATAAGTTCTCATATTCCT3600                           AsnIleAspHisPhePheSerSerProSerIleSerSerHisIlePro                               1185119011951200                                                               TCATTATCAATTTATTCTGCAATAGGTATAGAAACAGAAAATCTAGAT3648                           SerLeuSerIleTyrSerAlaIleGlyIleGluThrGluAsnLeuAsp                               120512101215                                                                   TTTTCAAAAAAAATAATGATGTTACCTAATGCTCCTTCAAGAGTGTTT3696                           PheSerLysLysIleMetMetLeuProAsnAlaProSerArgValPhe                               122012251230                                                                   TGGTGGGAAACTGGAGCAGTTCCAGGTTTAAGATCATTGGAAAATGAC3744                           TrpTrpGluThrGlyAlaValProGlyLeuArgSerLeuGluAsnAsp                               123512401245                                                                   GGAACTAGATTACTTGATTCAATAAGAGATTTATACCCAGGTAAATTT3792                           GlyThrArgLeuLeuAspSerIleArgAspLeuTyrProGlyLysPhe                               125012551260                                                                   TACTGGAGATTCTATGCTTTTTTCGATTATGCAATAACTACATTAAAA3840                           TyrTrpArgPheTyrAlaPhePheAspTyrAlaIleThrThrLeuLys                               1265127012751280                                                               CCAGTTTATGAAGACACTAATATTAAAATTAAACTAGATAAAGATACT3888                           ProValTyrGluAspThrAsnIleLysIleLysLeuAspLysAspThr                               128512901295                                                                   AGAAACTTCATAATGCCAACTATAACTACTAACGAAATTAGAAACAAA3936                           ArgAsnPheIleMetProThrIleThrThrAsnGluIleArgAsnLys                               130013051310                                                                   TTATCTTATTCATTTGATGGAGCAGGAGGAACTTACTCTTTATTATTA3984                           LeuSerTyrSerPheAspGlyAlaGlyGlyThrTyrSerLeuLeuLeu                               131513201325                                                                   TCTTCATATCCAATATCAACGAATATAAATTTATCTAAAGATGATTTA4032                           SerSerTyrProIleSerThrAsnIleAsnLeuSerLysAspAspLeu                               133013351340                                                                   TGGATATTTAATATTGATAATGAAGTAAGAGAAATATCTATAGAAAAT4080                           TrpIlePheAsnIleAspAsnGluValArgGluIleSerIleGluAsn                               1345135013551360                                                               GGTACTATTAAAAAAGGAAAGTTAATAAAAGATGTTTTAAGTAAAATT4128                           GlyThrIleLysLysGlyLysLeuIleLysAspValLeuSerLysIle                               136513701375                                                                   GATATAAATAAAAATAAACTTATTATAGGCAATCAAACAATAGATTTT4176                           AspIleAsnLysAsnLysLeuIleIleGlyAsnGlnThrIleAspPhe                               138013851390                                                                   TCAGGCGATATAGATAATAAAGATAGATATATATTCTTGACTTGTGAG4224                           SerGlyAspIleAspAsnLysAspArgTyrIlePheLeuThrCysGlu                               139514001405                                                                   TTAGATGATAAAATTAGTTTAATAATAGAAATAAATCTTGTTGCAAAA4272                           LeuAspAspLysIleSerLeuIleIleGluIleAsnLeuValAlaLys                               141014151420                                                                   TCTTATAGTTTGTTATTGTCTGGGGATAAAAATTATTTGATATCCAAT4320                           SerTyrSerLeuLeuLeuSerGlyAspLysAsnTyrLeuIleSerAsn                               1425143014351440                                                               TTATCTAATACTATTGAGAAAATCAATACTTTAGGCCTAGATAGTAAA4368                           LeuSerAsnThrIleGluLysIleAsnThrLeuGlyLeuAspSerLys                               144514501455                                                                   AATATAGCGTACAATTACACTGATGAATCTAATAATAAATATTTTGGA4416                           AsnIleAlaTyrAsnTyrThrAspGluSerAsnAsnLysTyrPheGly                               146014651470                                                                   GCTATATCTAAAACAAGTCAAAAAAGCATAATACATTATAAAAAAGAC4464                           AlaIleSerLysThrSerGlnLysSerIleIleHisTyrLysLysAsp                               147514801485                                                                   AGTAAAAATATATTAGAATTTTATAATGACAGTACATTAGAATTTAAC4512                           SerLysAsnIleLeuGluPheTyrAsnAspSerThrLeuGluPheAsn                               149014951500                                                                   AGTAAAGATTTTATTGCTGAAGATATAAATGTATTTATGAAAGATGAT4560                           SerLysAspPheIleAlaGluAspIleAsnValPheMetLysAspAsp                               1505151015151520                                                               ATTAATACTATAACAGGAAAATACTATGTTGATAATAATACTGATAAA4608                           IleAsnThrIleThrGlyLysTyrTyrValAspAsnAsnThrAspLys                               152515301535                                                                   AGTATAGATTTCTCTATTTCTTTAGTTAGTAAAAATCAAGTAAAAGTA4656                           SerIleAspPheSerIleSerLeuValSerLysAsnGlnValLysVal                               154015451550                                                                   AATGGATTATATTTAAATGAATCCGTATACTCATCTTACCTTGATTTT4704                           AsnGlyLeuTyrLeuAsnGluSerValTyrSerSerTyrLeuAspPhe                               155515601565                                                                   GTGAAAAATTCAGATGGACACCATAATACTTCTAATTTTATGAATTTA4752                           ValLysAsnSerAspGlyHisHisAsnThrSerAsnPheMetAsnLeu                               157015751580                                                                   TTTTTGGACAATATAAGTTTCTGGAAATTGTTTGGGTTTGAAAATATA4800                           PheLeuAspAsnIleSerPheTrpLysLeuPheGlyPheGluAsnIle                               1585159015951600                                                               AATTTTGTAATCGATAAATACTTTACCCTTGTTGGTAAAACTAATCTT4848                           AsnPheValIleAspLysTyrPheThrLeuValGlyLysThrAsnLeu                               160516101615                                                                   GGATATGTAGAATTTATTTGTGACAATAATAAAAATATAGATATATAT4896                           GlyTyrValGluPheIleCysAspAsnAsnLysAsnIleAspIleTyr                               162016251630                                                                   TTTGGTGAATGGAAAACATCGTCATCTAAAAGCACTATATTTAGCGGA4944                           PheGlyGluTrpLysThrSerSerSerLysSerThrIlePheSerGly                               163516401645                                                                   AATGGTAGAAATGTTGTAGTAGAGCCTATATATAATCCTGATACGGGT4992                           AsnGlyArgAsnValValValGluProIleTyrAsnProAspThrGly                               165016551660                                                                   GAAGATATATCTACTTCACTAGATTTTTCCTATGAACCTCTCTATGGA5040                           GluAspIleSerThrSerLeuAspPheSerTyrGluProLeuTyrGly                               1665167016751680                                                               ATAGATAGATATATAAATAAAGTATTGATAGCACCTGATTTATATACA5088                           IleAspArgTyrIleAsnLysValLeuIleAlaProAspLeuTyrThr                               168516901695                                                                   AGTTTAATAAATATTAATACCAATTATTATTCAAATGAGTACTACCCT5136                           SerLeuIleAsnIleAsnThrAsnTyrTyrSerAsnGluTyrTyrPro                               170017051710                                                                   GAGATTATAGTTCTTAACCCAAATACATTCCACAAAAAAGTAAATATA5184                           GluIleIleValLeuAsnProAsnThrPheHisLysLysValAsnIle                               171517201725                                                                   AATTTAGATAGTTCTTCTTTTGAGTATAAATGGTCTACAGAAGGAAGT5232                           AsnLeuAspSerSerSerPheGluTyrLysTrpSerThrGluGlySer                               173017351740                                                                   GACTTTATTTTAGTTAGATACTTAGAAGAAAGTAATAAAAAAATATTA5280                           AspPheIleLeuValArgTyrLeuGluGluSerAsnLysLysIleLeu                               1745175017551760                                                               CAAAAAATAAGAATCAAAGGTATCTTATCTAATACTCAATCATTTAAT5328                           GlnLysIleArgIleLysGlyIleLeuSerAsnThrGlnSerPheAsn                               176517701775                                                                   AAAATGAGTATAGATTTTAAAGATATTAAAAAACTATCATTAGGATAT5376                           LysMetSerIleAspPheLysAspIleLysLysLeuSerLeuGlyTyr                               178017851790                                                                   ATAATGAGTAATTTTAAATCATTTAATTCTGAAAATGAATTAGATAGA5424                           IleMetSerAsnPheLysSerPheAsnSerGluAsnGluLeuAspArg                               179518001805                                                                   GATCATTTAGGATTTAAAATAATAGATAATAAAACTTATTACTATGAT5472                           AspHisLeuGlyPheLysIleIleAspAsnLysThrTyrTyrTyrAsp                               181018151820                                                                   GAAGATAGTAAATTAGTTAAAGGATTAATCAATATAAATAATTCATTA5520                           GluAspSerLysLeuValLysGlyLeuIleAsnIleAsnAsnSerLeu                               1825183018351840                                                               TTCTATTTTGATCCTATAGAATTTAACTTAGTAACTGGATGGCAAACT5568                           PheTyrPheAspProIleGluPheAsnLeuValThrGlyTrpGlnThr                               184518501855                                                                   ATCAATGGTAAAAAATATTATTTTGATATAAATACTGGAGCAGCTTTA5616                           IleAsnGlyLysLysTyrTyrPheAspIleAsnThrGlyAlaAlaLeu                               186018651870                                                                   ACTAGTTATAAAATTATTAATGGTAAACACTTTTATTTTAATAATGAT5664                           ThrSerTyrLysIleIleAsnGlyLysHisPheTyrPheAsnAsnAsp                               187518801885                                                                   GGTGTGATGCAGTTGGGAGTATTTAAAGGACCTGATGGATTTGAATAT5712                           GlyValMetGlnLeuGlyValPheLysGlyProAspGlyPheGluTyr                               189018951900                                                                   TTTGCACCTGCCAATACTCAAAATAATAACATAGAAGGTCAGGCTATA5760                           PheAlaProAlaAsnThrGlnAsnAsnAsnIleGluGlyGlnAlaIle                               1905191019151920                                                               GTTTATCAAAGTAAATTCTTAACTTTGAATGGCAAAAAATATTATTTT5808                           ValTyrGlnSerLysPheLeuThrLeuAsnGlyLysLysTyrTyrPhe                               192519301935                                                                   GATAATAACTCAAAAGCAGTCACTGGATGGAGAATTATTAACAATGAG5856                           AspAsnAsnSerLysAlaValThrGlyTrpArgIleIleAsnAsnGlu                               194019451950                                                                   AAATATTACTTTAATCCTAATAATGCTATTGCTGCAGTCGGATTGCAA5904                           LysTyrTyrPheAsnProAsnAsnAlaIleAlaAlaValGlyLeuGln                               195519601965                                                                   GTAATTGACAATAATAAGTATTATTTCAATCCTGACACTGCTATCATC5952                           ValIleAspAsnAsnLysTyrTyrPheAsnProAspThrAlaIleIle                               197019751980                                                                   TCAAAAGGTTGGCAGACTGTTAATGGTAGTAGATACTACTTTGATACT6000                           SerLysGlyTrpGlnThrValAsnGlySerArgTyrTyrPheAspThr                               1985199019952000                                                               GATACCGCTATTGCCTTTAATGGTTATAAAACTATTGATGGTAAACAC6048                           AspThrAlaIleAlaPheAsnGlyTyrLysThrIleAspGlyLysHis                               200520102015                                                                   TTTTATTTTGATAGTGATTGTGTAGTGAAAATAGGTGTGTTTAGTACC6096                           PheTyrPheAspSerAspCysValValLysIleGlyValPheSerThr                               202020252030                                                                   TCTAATGGATTTGAATATTTTGCACCTGCTAATACTTATAATAATAAC6144                           SerAsnGlyPheGluTyrPheAlaProAlaAsnThrTyrAsnAsnAsn                               203520402045                                                                   ATAGAAGGTCAGGCTATAGTTTATCAAAGTAAATTCTTAACTTTGAAT6192                           IleGluGlyGlnAlaIleValTyrGlnSerLysPheLeuThrLeuAsn                               205020552060                                                                   GGTAAAAAATATTACTTTGATAATAACTCAAAAGCAGTTACCGGATTG6240                           GlyLysLysTyrTyrPheAspAsnAsnSerLysAlaValThrGlyLeu                               2065207020752080                                                               CAAACTATTGATAGTAAAAAATATTACTTTAATACTAACACTGCTGAA6288                           GlnThrIleAspSerLysLysTyrTyrPheAsnThrAsnThrAlaGlu                               208520902095                                                                   GCAGCTACTGGATGGCAAACTATTGATGGTAAAAAATATTACTTTAAT6336                           AlaAlaThrGlyTrpGlnThrIleAspGlyLysLysTyrTyrPheAsn                               210021052110                                                                   ACTAACACTGCTGAAGCAGCTACTGGATGGCAAACTATTGATGGTAAA6384                           ThrAsnThrAlaGluAlaAlaThrGlyTrpGlnThrIleAspGlyLys                               211521202125                                                                   AAATATTACTTTAATACTAACACTGCTATAGCTTCAACTGGTTATACA6432                           LysTyrTyrPheAsnThrAsnThrAlaIleAlaSerThrGlyTyrThr                               213021352140                                                                   ATTATTAATGGTAAACATTTTTATTTTAATACTGATGGTATTATGCAG6480                           IleIleAsnGlyLysHisPheTyrPheAsnThrAspGlyIleMetGln                               2145215021552160                                                               ATAGGAGTGTTTAAAGGACCTAATGGATTTGAATATTTTGCACCTGCT6528                           IleGlyValPheLysGlyProAsnGlyPheGluTyrPheAlaProAla                               216521702175                                                                   AATACGGATGCTAACAACATAGAAGGTCAAGCTATACTTTACCAAAAT6576                           AsnThrAspAlaAsnAsnIleGluGlyGlnAlaIleLeuTyrGlnAsn                               218021852190                                                                   GAATTCTTAACTTTGAATGGTAAAAAATATTACTTTGGTAGTGACTCA6624                           GluPheLeuThrLeuAsnGlyLysLysTyrTyrPheGlySerAspSer                               219522002205                                                                   AAAGCAGTTACTGGATGGAGAATTATTAACAATAAGAAATATTACTTT6672                           LysAlaValThrGlyTrpArgIleIleAsnAsnLysLysTyrTyrPhe                               221022152220                                                                   AATCCTAATAATGCTATTGCTGCAATTCATCTATGCACTATAAATAAT6720                           AsnProAsnAsnAlaIleAlaAlaIleHisLeuCysThrIleAsnAsn                               2225223022352240                                                               GACAAGTATTACTTTAGTTATGATGGAATTCTTCAAAATGGATATATT6768                           AspLysTyrTyrPheSerTyrAspGlyIleLeuGlnAsnGlyTyrIle                               224522502255                                                                   ACTATTGAAAGAAATAATTTCTATTTTGATGCTAATAATGAATCTAAA6816                           ThrIleGluArgAsnAsnPheTyrPheAspAlaAsnAsnGluSerLys                               226022652270                                                                   ATGGTAACAGGAGTATTTAAAGGACCTAATGGATTTGAGTATTTTGCA6864                           MetValThrGlyValPheLysGlyProAsnGlyPheGluTyrPheAla                               227522802285                                                                   CCTGCTAATACTCACAATAATAACATAGAAGGTCAGGCTATAGTTTAC6912                           ProAlaAsnThrHisAsnAsnAsnIleGluGlyGlnAlaIleValTyr                               229022952300                                                                   CAGAACAAATTCTTAACTTTGAATGGCAAAAAATATTATTTTGATAAT6960                           GlnAsnLysPheLeuThrLeuAsnGlyLysLysTyrTyrPheAspAsn                               2305231023152320                                                               GACTCAAAAGCAGTTACTGGATGGCAAACCATTGATGGTAAAAAATAT7008                           AspSerLysAlaValThrGlyTrpGlnThrIleAspGlyLysLysTyr                               232523302335                                                                   TACTTTAATCTTAACACTGCTGAAGCAGCTACTGGATGGCAAACTATT7056                           TyrPheAsnLeuAsnThrAlaGluAlaAlaThrGlyTrpGlnThrIle                               234023452350                                                                   GATGGTAAAAAATATTACTTTAATCTTAACACTGCTGAAGCAGCTACT7104                           AspGlyLysLysTyrTyrPheAsnLeuAsnThrAlaGluAlaAlaThr                               235523602365                                                                   GGATGGCAAACTATTGATGGTAAAAAATATTACTTTAATACTAACACT7152                           GlyTrpGlnThrIleAspGlyLysLysTyrTyrPheAsnThrAsnThr                               237023752380                                                                   TTCATAGCCTCAACTGGTTATACAAGTATTAATGGTAAACATTTTTAT7200                           PheIleAlaSerThrGlyTyrThrSerIleAsnGlyLysHisPheTyr                               2385239023952400                                                               TTTAATACTGATGGTATTATGCAGATAGGAGTGTTTAAAGGACCTAAT7248                           PheAsnThrAspGlyIleMetGlnIleGlyValPheLysGlyProAsn                               240524102415                                                                   GGATTTGAATACTTTGCACCTGCTAATACGGATGCTAACAACATAGAA7296                           GlyPheGluTyrPheAlaProAlaAsnThrAspAlaAsnAsnIleGlu                               242024252430                                                                   GGTCAAGCTATACTTTACCAAAATAAATTCTTAACTTTGAATGGTAAA7344                           GlyGlnAlaIleLeuTyrGlnAsnLysPheLeuThrLeuAsnGlyLys                               243524402445                                                                   AAATATTACTTTGGTAGTGACTCAAAAGCAGTTACCGGACTGCGAACT7392                           LysTyrTyrPheGlySerAspSerLysAlaValThrGlyLeuArgThr                               245024552460                                                                   ATTGATGGTAAAAAATATTACTTTAATACTAACACTGCTGTTGCAGTT7440                           IleAspGlyLysLysTyrTyrPheAsnThrAsnThrAlaValAlaVal                               2465247024752480                                                               ACTGGATGGCAAACTATTAATGGTAAAAAATACTACTTTAATACTAAC7488                           ThrGlyTrpGlnThrIleAsnGlyLysLysTyrTyrPheAsnThrAsn                               248524902495                                                                   ACTTCTATAGCTTCAACTGGTTATACAATTATTAGTGGTAAACATTTT7536                           ThrSerIleAlaSerThrGlyTyrThrIleIleSerGlyLysHisPhe                               250025052510                                                                   TATTTTAATACTGATGGTATTATGCAGATAGGAGTGTTTAAAGGACCT7584                           TyrPheAsnThrAspGlyIleMetGlnIleGlyValPheLysGlyPro                               251525202525                                                                   GATGGATTTGAATACTTTGCACCTGCTAATACAGATGCTAACAATATA7632                           AspGlyPheGluTyrPheAlaProAlaAsnThrAspAlaAsnAsnIle                               253025352540                                                                   GAAGGTCAAGCTATACGTTATCAAAATAGATTCCTATATTTACATGAC7680                           GluGlyGlnAlaIleArgTyrGlnAsnArgPheLeuTyrLeuHisAsp                               2545255025552560                                                               AATATATATTATTTTGGTAATAATTCAAAAGCGGCTACTGGTTGGGTA7728                           AsnIleTyrTyrPheGlyAsnAsnSerLysAlaAlaThrGlyTrpVal                               256525702575                                                                   ACTATTGATGGTAATAGATATTACTTCGAGCCTAATACAGCTATGGGT7776                           ThrIleAspGlyAsnArgTyrTyrPheGluProAsnThrAlaMetGly                               258025852590                                                                   GCGAATGGTTATAAAACTATTGATAATAAAAATTTTTACTTTAGAAAT7824                           AlaAsnGlyTyrLysThrIleAspAsnLysAsnPheTyrPheArgAsn                               259526002605                                                                   GGTTTACCTCAGATAGGAGTGTTTAAAGGGTCTAATGGATTTGAATAC7872                           GlyLeuProGlnIleGlyValPheLysGlySerAsnGlyPheGluTyr                               261026152620                                                                   TTTGCACCTGCTAATACGGATGCTAACAATATAGAAGGTCAAGCTATA7920                           PheAlaProAlaAsnThrAspAlaAsnAsnIleGluGlyGlnAlaIle                               2625263026352640                                                               CGTTATCAAAATAGATTCCTACATTTACTTGGAAAAATATATTACTTT7968                           ArgTyrGlnAsnArgPheLeuHisLeuLeuGlyLysIleTyrTyrPhe                               264526502655                                                                   GGTAATAATTCAAAAGCAGTTACTGGATGGCAAACTATTAATGGTAAA8016                           GlyAsnAsnSerLysAlaValThrGlyTrpGlnThrIleAsnGlyLys                               266026652670                                                                   GTATATTACTTTATGCCTGATACTGCTATGGCTGCAGCTGGTGGACTT8064                           ValTyrTyrPheMetProAspThrAlaMetAlaAlaAlaGlyGlyLeu                               267526802685                                                                   TTCGAGATTGATGGTGTTATATATTTCTTTGGTGTTGATGGAGTAAAA8112                           PheGluIleAspGlyValIleTyrPhePheGlyValAspGlyValLys                               269026952700                                                                   GCCCCTGGGATATATGGCTAA8133                                                      AlaProGlyIleTyrGly                                                             27052710                                                                       (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2710 amino acids                                                   (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        MetSerLeuIleSerLysGluGluLeuIleLysLeuAlaTyrSerIle                               151015                                                                         ArgProArgGluAsnGluTyrLysThrIleLeuThrAsnLeuAspGlu                               202530                                                                         TyrAsnLysLeuThrThrAsnAsnAsnGluAsnLysTyrLeuGlnLeu                               354045                                                                         LysLysLeuAsnGluSerIleAspValPheMetAsnLysTyrLysThr                               505560                                                                         SerSerArgAsnArgAlaLeuSerAsnLeuLysLysAspIleLeuLys                               65707580                                                                       GluValIleLeuIleLysAsnSerAsnThrSerProValGluLysAsn                               859095                                                                         LeuHisPheValTrpIleGlyGlyGluValSerAspIleAlaLeuGlu                               100105110                                                                      TyrIleLysGlnTrpAlaAspIleAsnAlaGluTyrAsnIleLysLeu                               115120125                                                                      TrpTyrAspSerGluAlaPheLeuValAsnThrLeuLysLysAlaIle                               130135140                                                                      ValGluSerSerThrThrGluAlaLeuGlnLeuLeuGluGluGluIle                               145150155160                                                                   GlnAsnProGlnPheAspAsnMetLysPheTyrLysLysArgMetGlu                               165170175                                                                      PheIleTyrAspArgGlnLysArgPheIleAsnTyrTyrLysSerGln                               180185190                                                                      IleAsnLysProThrValProThrIleAspAspIleIleLysSerHis                               195200205                                                                      LeuValSerGluTyrAsnArgAspGluThrValLeuGluSerTyrArg                               210215220                                                                      ThrAsnSerLeuArgLysIleAsnSerAsnHisGlyIleAspIleArg                               225230235240                                                                   AlaAsnSerLeuPheThrGluGlnGluLeuLeuAsnIleTyrSerGln                               245250255                                                                      GluLeuLeuAsnArgGlyAsnLeuAlaAlaAlaSerAspIleValArg                               260265270                                                                      LeuLeuAlaLeuLysAsnPheGlyGlyValTyrLeuAspValAspMet                               275280285                                                                      LeuProGlyIleHisSerAspLeuPheLysThrIleSerArgProSer                               290295300                                                                      SerIleGlyLeuAspArgTrpGluMetIleLysLeuGluAlaIleMet                               305310315320                                                                   LysTyrLysLysTyrIleAsnAsnTyrThrSerGluAsnPheAspLys                               325330335                                                                      LeuAspGlnGlnLeuLysAspAsnPheLysLeuIleIleGluSerLys                               340345350                                                                      SerGluLysSerGluIlePheSerLysLeuGluAsnLeuAsnValSer                               355360365                                                                      AspLeuGluIleLysIleAlaPheAlaLeuGlySerValIleAsnGln                               370375380                                                                      AlaLeuIleSerLysGlnGlySerTyrLeuThrAsnLeuValIleGlu                               385390395400                                                                   GlnValLysAsnArgTyrGlnPheLeuAsnGlnHisLeuAsnProAla                               405410415                                                                      IleGluSerAspAsnAsnPheThrAspThrThrLysIlePheHisAsp                               420425430                                                                      SerLeuPheAsnSerAlaThrAlaGluAsnSerMetPheLeuThrLys                               435440445                                                                      IleAlaProTyrLeuGlnValGlyPheMetProGluAlaArgSerThr                               450455460                                                                      IleSerLeuSerGlyProGlyAlaTyrAlaSerAlaTyrTyrAspPhe                               465470475480                                                                   IleAsnLeuGlnGluAsnThrIleGluLysThrLeuLysAlaSerAsp                               485490495                                                                      LeuIleGluPheLysPheProGluAsnAsnLeuSerGlnLeuThrGlu                               500505510                                                                      GlnGluIleAsnSerLeuTrpSerPheAspGlnAlaSerAlaLysTyr                               515520525                                                                      GlnPheGluLysTyrValArgAspTyrThrGlyGlySerLeuSerGlu                               530535540                                                                      AspAsnGlyValAspPheAsnLysAsnThrAlaLeuAspLysAsnTyr                               545550555560                                                                   LeuLeuAsnAsnLysIleProSerAsnAsnValGluGluAlaGlySer                               565570575                                                                      LysAsnTyrValHisTyrIleIleGlnLeuGlnGlyAspAspIleSer                               580585590                                                                      TyrGluAlaThrCysAsnLeuPheSerLysAsnProLysAsnSerIle                               595600605                                                                      IleIleGlnArgAsnMetAsnGluSerAlaLysSerTyrPheLeuSer                               610615620                                                                      AspAspGlyGluSerIleLeuGluLeuAsnLysTyrArgIleProGlu                               625630635640                                                                   ArgLeuLysAsnLysGluLysValLysValThrPheIleGlyHisGly                               645650655                                                                      LysAspGluPheAsnThrSerGluPheAlaArgLeuSerValAspSer                               660665670                                                                      LeuSerAsnGluIleSerSerPheLeuAspThrIleLysLeuAspIle                               675680685                                                                      SerProLysAsnValGluValAsnLeuLeuGlyCysAsnMetPheSer                               690695700                                                                      TyrAspPheAsnValGluGluThrTyrProGlyLysLeuLeuLeuSer                               705710715720                                                                   IleMetAspLysIleThrSerThrLeuProAspValAsnLysAsnSer                               725730735                                                                      IleThrIleGlyAlaAsnGlnTyrGluValArgIleAsnSerGluGly                               740745750                                                                      ArgLysGluLeuLeuAlaHisSerGlyLysTrpIleAsnLysGluGlu                               755760765                                                                      AlaIleMetSerAspLeuSerSerLysGluTyrIlePhePheAspSer                               770775780                                                                      IleAspAsnLysLeuLysAlaLysSerLysAsnIleProGlyLeuAla                               785790795800                                                                   SerIleSerGluAspIleLysThrLeuLeuLeuAspAlaSerValSer                               805810815                                                                      ProAspThrLysPheIleLeuAsnAsnLeuLysLeuAsnIleGluSer                               820825830                                                                      SerIleGlyAspTyrIleTyrTyrGluLysLeuGluProValLysAsn                               835840845                                                                      IleIleHisAsnSerIleAspAspLeuIleAspGluPheAsnLeuLeu                               850855860                                                                      GluAsnValSerAspGluLeuTyrGluLeuLysLysLeuAsnAsnLeu                               865870875880                                                                   AspGluLysTyrLeuIleSerPheGluAspIleSerLysAsnAsnSer                               885890895                                                                      ThrTyrSerValArgPheIleAsnLysSerAsnGlyGluSerValTyr                               900905910                                                                      ValGluThrGluLysGluIlePheSerLysTyrSerGluHisIleThr                               915920925                                                                      LysGluIleSerThrIleLysAsnSerIleIleThrAspValAsnGly                               930935940                                                                      AsnLeuLeuAspAsnIleGlnLeuAspHisThrSerGlnValAsnThr                               945950955960                                                                   LeuAsnAlaAlaPhePheIleGlnSerLeuIleAspTyrSerSerAsn                               965970975                                                                      LysAspValLeuAsnAspLeuSerThrSerValLysValGlnLeuTyr                               980985990                                                                      AlaGlnLeuPheSerThrGlyLeuAsnThrIleTyrAspSerIleGln                               99510001005                                                                    LeuValAsnLeuIleSerAsnAlaValAsnAspThrIleAsnValLeu                               101010151020                                                                   ProThrIleThrGluGlyIleProIleValSerThrIleLeuAspGly                               1025103010351040                                                               IleAsnLeuGlyAlaAlaIleLysGluLeuLeuAspGluHisAspPro                               104510501055                                                                   LeuLeuLysLysGluLeuGluAlaLysValGlyValLeuAlaIleAsn                               106010651070                                                                   MetSerLeuSerIleAlaAlaThrValAlaSerIleValGlyIleGly                               107510801085                                                                   AlaGluValThrIlePheLeuLeuProIleAlaGlyIleSerAlaGly                               109010951100                                                                   IleProSerLeuValAsnAsnGluLeuIleLeuHisAspLysAlaThr                               1105111011151120                                                               SerValValAsnTyrPheAsnHisLeuSerGluSerLysLysTyrGly                               112511301135                                                                   ProLeuLysThrGluAspAspLysIleLeuValProIleAspAspLeu                               114011451150                                                                   ValIleSerGluIleAspPheAsnAsnAsnSerIleLysLeuGlyThr                               115511601165                                                                   CysAsnIleLeuAlaMetGluGlyGlySerGlyHisThrValThrGly                               117011751180                                                                   AsnIleAspHisPhePheSerSerProSerIleSerSerHisIlePro                               1185119011951200                                                               SerLeuSerIleTyrSerAlaIleGlyIleGluThrGluAsnLeuAsp                               120512101215                                                                   PheSerLysLysIleMetMetLeuProAsnAlaProSerArgValPhe                               122012251230                                                                   TrpTrpGluThrGlyAlaValProGlyLeuArgSerLeuGluAsnAsp                               123512401245                                                                   GlyThrArgLeuLeuAspSerIleArgAspLeuTyrProGlyLysPhe                               125012551260                                                                   TyrTrpArgPheTyrAlaPhePheAspTyrAlaIleThrThrLeuLys                               1265127012751280                                                               ProValTyrGluAspThrAsnIleLysIleLysLeuAspLysAspThr                               128512901295                                                                   ArgAsnPheIleMetProThrIleThrThrAsnGluIleArgAsnLys                               130013051310                                                                   LeuSerTyrSerPheAspGlyAlaGlyGlyThrTyrSerLeuLeuLeu                               131513201325                                                                   SerSerTyrProIleSerThrAsnIleAsnLeuSerLysAspAspLeu                               133013351340                                                                   TrpIlePheAsnIleAspAsnGluValArgGluIleSerIleGluAsn                               1345135013551360                                                               GlyThrIleLysLysGlyLysLeuIleLysAspValLeuSerLysIle                               136513701375                                                                   AspIleAsnLysAsnLysLeuIleIleGlyAsnGlnThrIleAspPhe                               138013851390                                                                   SerGlyAspIleAspAsnLysAspArgTyrIlePheLeuThrCysGlu                               139514001405                                                                   LeuAspAspLysIleSerLeuIleIleGluIleAsnLeuValAlaLys                               141014151420                                                                   SerTyrSerLeuLeuLeuSerGlyAspLysAsnTyrLeuIleSerAsn                               1425143014351440                                                               LeuSerAsnThrIleGluLysIleAsnThrLeuGlyLeuAspSerLys                               144514501455                                                                   AsnIleAlaTyrAsnTyrThrAspGluSerAsnAsnLysTyrPheGly                               146014651470                                                                   AlaIleSerLysThrSerGlnLysSerIleIleHisTyrLysLysAsp                               147514801485                                                                   SerLysAsnIleLeuGluPheTyrAsnAspSerThrLeuGluPheAsn                               149014951500                                                                   SerLysAspPheIleAlaGluAspIleAsnValPheMetLysAspAsp                               1505151015151520                                                               IleAsnThrIleThrGlyLysTyrTyrValAspAsnAsnThrAspLys                               152515301535                                                                   SerIleAspPheSerIleSerLeuValSerLysAsnGlnValLysVal                               154015451550                                                                   AsnGlyLeuTyrLeuAsnGluSerValTyrSerSerTyrLeuAspPhe                               155515601565                                                                   ValLysAsnSerAspGlyHisHisAsnThrSerAsnPheMetAsnLeu                               157015751580                                                                   PheLeuAspAsnIleSerPheTrpLysLeuPheGlyPheGluAsnIle                               1585159015951600                                                               AsnPheValIleAspLysTyrPheThrLeuValGlyLysThrAsnLeu                               160516101615                                                                   GlyTyrValGluPheIleCysAspAsnAsnLysAsnIleAspIleTyr                               162016251630                                                                   PheGlyGluTrpLysThrSerSerSerLysSerThrIlePheSerGly                               163516401645                                                                   AsnGlyArgAsnValValValGluProIleTyrAsnProAspThrGly                               165016551660                                                                   GluAspIleSerThrSerLeuAspPheSerTyrGluProLeuTyrGly                               1665167016751680                                                               IleAspArgTyrIleAsnLysValLeuIleAlaProAspLeuTyrThr                               168516901695                                                                   SerLeuIleAsnIleAsnThrAsnTyrTyrSerAsnGluTyrTyrPro                               170017051710                                                                   GluIleIleValLeuAsnProAsnThrPheHisLysLysValAsnIle                               171517201725                                                                   AsnLeuAspSerSerSerPheGluTyrLysTrpSerThrGluGlySer                               173017351740                                                                   AspPheIleLeuValArgTyrLeuGluGluSerAsnLysLysIleLeu                               1745175017551760                                                               GlnLysIleArgIleLysGlyIleLeuSerAsnThrGlnSerPheAsn                               176517701775                                                                   LysMetSerIleAspPheLysAspIleLysLysLeuSerLeuGlyTyr                               178017851790                                                                   IleMetSerAsnPheLysSerPheAsnSerGluAsnGluLeuAspArg                               179518001805                                                                   AspHisLeuGlyPheLysIleIleAspAsnLysThrTyrTyrTyrAsp                               181018151820                                                                   GluAspSerLysLeuValLysGlyLeuIleAsnIleAsnAsnSerLeu                               1825183018351840                                                               PheTyrPheAspProIleGluPheAsnLeuValThrGlyTrpGlnThr                               184518501855                                                                   IleAsnGlyLysLysTyrTyrPheAspIleAsnThrGlyAlaAlaLeu                               186018651870                                                                   ThrSerTyrLysIleIleAsnGlyLysHisPheTyrPheAsnAsnAsp                               187518801885                                                                   GlyValMetGlnLeuGlyValPheLysGlyProAspGlyPheGluTyr                               189018951900                                                                   PheAlaProAlaAsnThrGlnAsnAsnAsnIleGluGlyGlnAlaIle                               1905191019151920                                                               ValTyrGlnSerLysPheLeuThrLeuAsnGlyLysLysTyrTyrPhe                               192519301935                                                                   AspAsnAsnSerLysAlaValThrGlyTrpArgIleIleAsnAsnGlu                               194019451950                                                                   LysTyrTyrPheAsnProAsnAsnAlaIleAlaAlaValGlyLeuGln                               195519601965                                                                   ValIleAspAsnAsnLysTyrTyrPheAsnProAspThrAlaIleIle                               197019751980                                                                   SerLysGlyTrpGlnThrValAsnGlySerArgTyrTyrPheAspThr                               1985199019952000                                                               AspThrAlaIleAlaPheAsnGlyTyrLysThrIleAspGlyLysHis                               200520102015                                                                   PheTyrPheAspSerAspCysValValLysIleGlyValPheSerThr                               202020252030                                                                   SerAsnGlyPheGluTyrPheAlaProAlaAsnThrTyrAsnAsnAsn                               203520402045                                                                   IleGluGlyGlnAlaIleValTyrGlnSerLysPheLeuThrLeuAsn                               205020552060                                                                   GlyLysLysTyrTyrPheAspAsnAsnSerLysAlaValThrGlyLeu                               2065207020752080                                                               GlnThrIleAspSerLysLysTyrTyrPheAsnThrAsnThrAlaGlu                               208520902095                                                                   AlaAlaThrGlyTrpGlnThrIleAspGlyLysLysTyrTyrPheAsn                               210021052110                                                                   ThrAsnThrAlaGluAlaAlaThrGlyTrpGlnThrIleAspGlyLys                               211521202125                                                                   LysTyrTyrPheAsnThrAsnThrAlaIleAlaSerThrGlyTyrThr                               213021352140                                                                   IleIleAsnGlyLysHisPheTyrPheAsnThrAspGlyIleMetGln                               2145215021552160                                                               IleGlyValPheLysGlyProAsnGlyPheGluTyrPheAlaProAla                               216521702175                                                                   AsnThrAspAlaAsnAsnIleGluGlyGlnAlaIleLeuTyrGlnAsn                               218021852190                                                                   GluPheLeuThrLeuAsnGlyLysLysTyrTyrPheGlySerAspSer                               219522002205                                                                   LysAlaValThrGlyTrpArgIleIleAsnAsnLysLysTyrTyrPhe                               221022152220                                                                   AsnProAsnAsnAlaIleAlaAlaIleHisLeuCysThrIleAsnAsn                               2225223022352240                                                               AspLysTyrTyrPheSerTyrAspGlyIleLeuGlnAsnGlyTyrIle                               224522502255                                                                   ThrIleGluArgAsnAsnPheTyrPheAspAlaAsnAsnGluSerLys                               226022652270                                                                   MetValThrGlyValPheLysGlyProAsnGlyPheGluTyrPheAla                               227522802285                                                                   ProAlaAsnThrHisAsnAsnAsnIleGluGlyGlnAlaIleValTyr                               229022952300                                                                   GlnAsnLysPheLeuThrLeuAsnGlyLysLysTyrTyrPheAspAsn                               2305231023152320                                                               AspSerLysAlaValThrGlyTrpGlnThrIleAspGlyLysLysTyr                               232523302335                                                                   TyrPheAsnLeuAsnThrAlaGluAlaAlaThrGlyTrpGlnThrIle                               234023452350                                                                   AspGlyLysLysTyrTyrPheAsnLeuAsnThrAlaGluAlaAlaThr                               235523602365                                                                   GlyTrpGlnThrIleAspGlyLysLysTyrTyrPheAsnThrAsnThr                               237023752380                                                                   PheIleAlaSerThrGlyTyrThrSerIleAsnGlyLysHisPheTyr                               2385239023952400                                                               PheAsnThrAspGlyIleMetGlnIleGlyValPheLysGlyProAsn                               240524102415                                                                   GlyPheGluTyrPheAlaProAlaAsnThrAspAlaAsnAsnIleGlu                               242024252430                                                                   GlyGlnAlaIleLeuTyrGlnAsnLysPheLeuThrLeuAsnGlyLys                               243524402445                                                                   LysTyrTyrPheGlySerAspSerLysAlaValThrGlyLeuArgThr                               245024552460                                                                   IleAspGlyLysLysTyrTyrPheAsnThrAsnThrAlaValAlaVal                               2465247024752480                                                               ThrGlyTrpGlnThrIleAsnGlyLysLysTyrTyrPheAsnThrAsn                               248524902495                                                                   ThrSerIleAlaSerThrGlyTyrThrIleIleSerGlyLysHisPhe                               250025052510                                                                   TyrPheAsnThrAspGlyIleMetGlnIleGlyValPheLysGlyPro                               251525202525                                                                   AspGlyPheGluTyrPheAlaProAlaAsnThrAspAlaAsnAsnIle                               253025352540                                                                   GluGlyGlnAlaIleArgTyrGlnAsnArgPheLeuTyrLeuHisAsp                               2545255025552560                                                               AsnIleTyrTyrPheGlyAsnAsnSerLysAlaAlaThrGlyTrpVal                               256525702575                                                                   ThrIleAspGlyAsnArgTyrTyrPheGluProAsnThrAlaMetGly                               258025852590                                                                   AlaAsnGlyTyrLysThrIleAspAsnLysAsnPheTyrPheArgAsn                               259526002605                                                                   GlyLeuProGlnIleGlyValPheLysGlySerAsnGlyPheGluTyr                               261026152620                                                                   PheAlaProAlaAsnThrAspAlaAsnAsnIleGluGlyGlnAlaIle                               2625263026352640                                                               ArgTyrGlnAsnArgPheLeuHisLeuLeuGlyLysIleTyrTyrPhe                               264526502655                                                                   GlyAsnAsnSerLysAlaValThrGlyTrpGlnThrIleAsnGlyLys                               266026652670                                                                   ValTyrTyrPheMetProAspThrAlaMetAlaAlaAlaGlyGlyLeu                               267526802685                                                                   PheGluIleAspGlyValIleTyrPhePheGlyValAspGlyValLys                               269026952700                                                                   AlaProGlyIleTyrGly                                                             27052710                                                                       (2) INFORMATION FOR SEQ ID NO:7:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 811 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: unknown                                                      (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        SerTyrLysIleIleAsnGlyLysHisPheTyrPheAsnAsnAspGly                               151015                                                                         ValMetGlnLeuGlyValPheLysGlyProAspGlyPheGluTyrPhe                               202530                                                                         AlaProAlaAsnThrGlnAsnAsnAsnIleGluGlyGlnAlaIleVal                               354045                                                                         TyrGlnSerLysPheLeuThrLeuAsnGlyLysLysTyrTyrPheAsp                               505560                                                                         AsnAsnSerLysAlaValThrGlyTrpArgIleIleAsnAsnGluLys                               65707580                                                                       TyrTyrPheAsnProAsnAsnAlaIleAlaAlaValGlyLeuGlnVal                               859095                                                                         IleAspAsnAsnLysTyrTyrPheAsnProAspThrAlaIleIleSer                               100105110                                                                      LysGlyTrpGlnThrValAsnGlySerArgTyrTyrPheAspThrAsp                               115120125                                                                      ThrAlaIleAlaPheAsnGlyTyrLysThrIleAspGlyLysHisPhe                               130135140                                                                      TyrPheAspSerAspCysValValLysIleGlyValPheSerThrSer                               145150155160                                                                   AsnGlyPheGluTyrPheAlaProAlaAsnThrTyrAsnAsnAsnIle                               165170175                                                                      GluGlyGlnAlaIleValTyrGlnSerLysPheLeuThrLeuAsnGly                               180185190                                                                      LysLysTyrTyrPheAspAsnAsnSerLysAlaValThrGlyLeuGln                               195200205                                                                      ThrIleAspSerLysLysTyrTyrPheAsnThrAsnThrAlaGluAla                               210215220                                                                      AlaThrGlyTrpGlnThrIleAspGlyLysLysTyrTyrPheAsnThr                               225230235240                                                                   AsnThrAlaGluAlaAlaThrGlyTrpGlnThrIleAspGlyLysLys                               245250255                                                                      TyrTyrPheAsnThrAsnThrAlaIleAlaSerThrGlyTyrThrIle                               260265270                                                                      IleAsnGlyLysHisPheTyrPheAsnThrAspGlyIleMetGlnIle                               275280285                                                                      GlyValPheLysGlyProAsnGlyPheGluTyrPheAlaProAlaAsn                               290295300                                                                      ThrAspAlaAsnAsnIleGluGlyGlnAlaIleLeuTyrGlnAsnGlu                               305310315320                                                                   PheLeuThrLeuAsnGlyLysLysTyrTyrPheGlySerAspSerLys                               325330335                                                                      AlaValThrGlyTrpArgIleIleAsnAsnLysLysTyrTyrPheAsn                               340345350                                                                      ProAsnAsnAlaIleAlaAlaIleHisLeuCysThrIleAsnAsnAsp                               355360365                                                                      LysTyrTyrPheSerTyrAspGlyIleLeuGlnAsnGlyTyrIleThr                               370375380                                                                      IleGluArgAsnAsnPheTyrPheAspAlaAsnAsnGluSerLysMet                               385390395400                                                                   ValThrGlyValPheLysGlyProAsnGlyPheGluTyrPheAlaPro                               405410415                                                                      AlaAsnThrHisAsnAsnAsnIleGluGlyGlnAlaIleValTyrGln                               420425430                                                                      AsnLysPheLeuThrLeuAsnGlyLysLysTyrTyrPheAspAsnAsp                               435440445                                                                      SerLysAlaValThrGlyTrpGlnThrIleAspGlyLysLysTyrTyr                               450455460                                                                      PheAsnLeuAsnThrAlaGluAlaAlaThrGlyTrpGlnThrIleAsp                               465470475480                                                                   GlyLysLysTyrTyrPheAsnLeuAsnThrAlaGluAlaAlaThrGly                               485490495                                                                      TrpGlnThrIleAspGlyLysLysTyrTyrPheAsnThrAsnThrPhe                               500505510                                                                      IleAlaSerThrGlyTyrThrSerIleAsnGlyLysHisPheTyrPhe                               515520525                                                                      AsnThrAspGlyIleMetGlnIleGlyValPheLysGlyProAsnGly                               530535540                                                                      PheGluTyrPheAlaProAlaAsnThrAspAlaAsnAsnIleGluGly                               545550555560                                                                   GlnAlaIleLeuTyrGlnAsnLysPheLeuThrLeuAsnGlyLysLys                               565570575                                                                      TyrTyrPheGlySerAspSerLysAlaValThrGlyLeuArgThrIle                               580585590                                                                      AspGlyLysLysTyrTyrPheAsnThrAsnThrAlaValAlaValThr                               595600605                                                                      GlyTrpGlnThrIleAsnGlyLysLysTyrTyrPheAsnThrAsnThr                               610615620                                                                      SerIleAlaSerThrGlyTyrThrIleIleSerGlyLysHisPheTyr                               625630635640                                                                   PheAsnThrAspGlyIleMetGlnIleGlyValPheLysGlyProAsp                               645650655                                                                      GlyPheGluTyrPheAlaProAlaAsnThrAspAlaAsnAsnIleGlu                               660665670                                                                      GlyGlnAlaIleArgTyrGlnAsnArgPheLeuTyrLeuHisAspAsn                               675680685                                                                      IleTyrTyrPheGlyAsnAsnSerLysAlaAlaThrGlyTrpValThr                               690695700                                                                      IleAspGlyAsnArgTyrTyrPheGluProAsnThrAlaMetGlyAla                               705710715720                                                                   AsnGlyTyrLysThrIleAspAsnLysAsnPheTyrPheArgAsnGly                               725730735                                                                      LeuProGlnIleGlyValPheLysGlySerAsnGlyPheGluTyrPhe                               740745750                                                                      AlaProAlaAsnThrAspAlaAsnAsnIleGluGlyGlnAlaIleArg                               755760765                                                                      TyrGlnAsnArgPheLeuHisLeuLeuGlyLysIleTyrTyrPheGly                               770775780                                                                      AsnAsnSerLysAlaValThrGlyTrpGlnThrIleAsnGlyLysVal                               785790795800                                                                   TyrTyrPheMetProAspThrAlaMetAlaAla                                              805810                                                                         (2) INFORMATION FOR SEQ ID NO:8:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 91 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: unknown                                                      (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                        SerTyrLysIleIleAsnGlyLysHisPheTyrPheAsnAsnAspGly                               151015                                                                         ValMetGlnLeuGlyValPheLysGlyProAspGlyPheGluTyrPhe                               202530                                                                         AlaProAlaAsnThrGlnAsnAsnAsnIleGluGlyGlnAlaIleVal                               354045                                                                         TyrGlnSerLysPheLeuThrLeuAsnGlyLysLysTyrTyrPheAsp                               505560                                                                         AsnAsnSerLysAlaValThrGlyTrpArgIleIleAsnAsnGluLys                               65707580                                                                       TyrTyrPheAsnProAsnAsnAlaIleAlaAla                                              8590                                                                           (2) INFORMATION FOR SEQ ID NO:9:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 7101 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..7098                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                        ATGAGTTTAGTTAATAGAAAACAGTTAGAAAAAATGGCAAATGTAAGA48                             MetSerLeuValAsnArgLysGlnLeuGluLysMetAlaAsnValArg                               151015                                                                         TTTCGTACTCAAGAAGATGAATATGTTGCAATATTGGATGCTTTAGAA96                             PheArgThrGlnGluAspGluTyrValAlaIleLeuAspAlaLeuGlu                               202530                                                                         GAATATCATAATATGTCAGAGAATACTGTAGTCGAAAAATATTTAAAA144                            GluTyrHisAsnMetSerGluAsnThrValValGluLysTyrLeuLys                               354045                                                                         TTAAAAGATATAAATAGTTTAACAGATATTTATATAGATACATATAAA192                            LeuLysAspIleAsnSerLeuThrAspIleTyrIleAspThrTyrLys                               505560                                                                         AAATCTGGTAGAAATAAAGCCTTAAAAAAATTTAAGGAATATCTAGTT240                            LysSerGlyArgAsnLysAlaLeuLysLysPheLysGluTyrLeuVal                               65707580                                                                       ACAGAAGTATTAGAGCTAAAGAATAATAATTTAACTCCAGTTGAGAAA288                            ThrGluValLeuGluLeuLysAsnAsnAsnLeuThrProValGluLys                               859095                                                                         AATTTACATTTTGTTTGGATTGGAGGTCAAATAAATGACACTGCTATT336                            AsnLeuHisPheValTrpIleGlyGlyGlnIleAsnAspThrAlaIle                               100105110                                                                      AATTATATAAATCAATGGAAAGATGTAAATAGTGATTATAATGTTAAT384                            AsnTyrIleAsnGlnTrpLysAspValAsnSerAspTyrAsnValAsn                               115120125                                                                      GTTTTTTATGATAGTAATGCATTTTTGATAAACACATTGAAAAAAACT432                            ValPheTyrAspSerAsnAlaPheLeuIleAsnThrLeuLysLysThr                               130135140                                                                      GTAGTAGAATCAGCAATAAATGATACACTTGAATCATTTAGAGAAAAC480                            ValValGluSerAlaIleAsnAspThrLeuGluSerPheArgGluAsn                               145150155160                                                                   TTAAATGACCCTAGATTTGACTATAATAAATTCTTCAGAAAACGTATG528                            LeuAsnAspProArgPheAspTyrAsnLysPhePheArgLysArgMet                               165170175                                                                      GAAATAATTTATGATAAACAGAAAAATTTCATAAACTACTATAAAGCT576                            GluIleIleTyrAspLysGlnLysAsnPheIleAsnTyrTyrLysAla                               180185190                                                                      CAAAGAGAAGAAAATCCTGAACTTATAATTGATGATATTGTAAAGACA624                            GlnArgGluGluAsnProGluLeuIleIleAspAspIleValLysThr                               195200205                                                                      TATCTTTCAAATGAGTATTCAAAGGAGATAGATGAACTTAATACCTAT672                            TyrLeuSerAsnGluTyrSerLysGluIleAspGluLeuAsnThrTyr                               210215220                                                                      ATTGAAGAATCCTTAAATAAAATTACACAGAATAGTGGAAATGATGTT720                            IleGluGluSerLeuAsnLysIleThrGlnAsnSerGlyAsnAspVal                               225230235240                                                                   AGAAACTTTGAAGAATTTAAAAATGGAGAGTCATTCAACTTATATGAA768                            ArgAsnPheGluGluPheLysAsnGlyGluSerPheAsnLeuTyrGlu                               245250255                                                                      CAAGAGTTGGTAGAAAGGTGGAATTTAGCTGCTGCTTCTGACATATTA816                            GlnGluLeuValGluArgTrpAsnLeuAlaAlaAlaSerAspIleLeu                               260265270                                                                      AGAATATCTGCATTAAAAGAAATTGGTGGTATGTATTTAGATGTTGAT864                            ArgIleSerAlaLeuLysGluIleGlyGlyMetTyrLeuAspValAsp                               275280285                                                                      ATGTTACCAGGAATACAACCAGACTTATTTGAGTCTATAGAGAAACCT912                            MetLeuProGlyIleGlnProAspLeuPheGluSerIleGluLysPro                               290295300                                                                      AGTTCAGTAACAGTGGATTTTTGGGAAATGACAAAGTTAGAAGCTATA960                            SerSerValThrValAspPheTrpGluMetThrLysLeuGluAlaIle                               305310315320                                                                   ATGAAATACAAAGAATATATACCAGAATATACCTCAGAACATTTTGAC1008                           MetLysTyrLysGluTyrIleProGluTyrThrSerGluHisPheAsp                               325330335                                                                      ATGTTAGACGAAGAAGTTCAAAGTAGTTTTGAATCTGTTCTAGCTTCT1056                           MetLeuAspGluGluValGlnSerSerPheGluSerValLeuAlaSer                               340345350                                                                      AAGTCAGATAAATCAGAAATATTCTCATCACTTGGTGATATGGAGGCA1104                           LysSerAspLysSerGluIlePheSerSerLeuGlyAspMetGluAla                               355360365                                                                      TCACCACTAGAAGTTAAAATTGCATTTAATAGTAAGGGTATTATAAAT1152                           SerProLeuGluValLysIleAlaPheAsnSerLysGlyIleIleAsn                               370375380                                                                      CAAGGGCTAATTTCTGTGAAAGACTCATATTGTAGCAATTTAATAGTA1200                           GlnGlyLeuIleSerValLysAspSerTyrCysSerAsnLeuIleVal                               385390395400                                                                   AAACAAATCGAGAATAGATATAAAATATTGAATAATAGTTTAAATCCA1248                           LysGlnIleGluAsnArgTyrLysIleLeuAsnAsnSerLeuAsnPro                               405410415                                                                      GCTATTAGCGAGGATAATGATTTTAATACTACAACGAATACCTTTATT1296                           AlaIleSerGluAspAsnAspPheAsnThrThrThrAsnThrPheIle                               420425430                                                                      GATAGTATAATGGCTGAAGCTAATGCAGATAATGGTAGATTTATGATG1344                           AspSerIleMetAlaGluAlaAsnAlaAspAsnGlyArgPheMetMet                               435440445                                                                      GAACTAGGAAAGTATTTAAGAGTTGGTTTCTTCCCAGATGTTAAAACT1392                           GluLeuGlyLysTyrLeuArgValGlyPhePheProAspValLysThr                               450455460                                                                      ACTATTAACTTAAGTGGCCCTGAAGCATATGCGGCAGCTTATCAAGAT1440                           ThrIleAsnLeuSerGlyProGluAlaTyrAlaAlaAlaTyrGlnAsp                               465470475480                                                                   TTATTAATGTTTAAAGAAGGCAGTATGAATATCCATTTGATAGAAGCT1488                           LeuLeuMetPheLysGluGlySerMetAsnIleHisLeuIleGluAla                               485490495                                                                      GATTTAAGAAACTTTGAAATCTCTAAAACTAATATTTCTCAATCAACT1536                           AspLeuArgAsnPheGluIleSerLysThrAsnIleSerGlnSerThr                               500505510                                                                      GAACAAGAAATGGCTAGCTTATGGTCATTTGACGATGCAAGAGCTAAA1584                           GluGlnGluMetAlaSerLeuTrpSerPheAspAspAlaArgAlaLys                               515520525                                                                      GCTCAATTTGAAGAATATAAAAGGAATTATTTTGAAGGTTCTCTTGGT1632                           AlaGlnPheGluGluTyrLysArgAsnTyrPheGluGlySerLeuGly                               530535540                                                                      GAAGATGATAATCTTGATTTTTCTCAAAATATAGTAGTTGACAAGGAG1680                           GluAspAspAsnLeuAspPheSerGlnAsnIleValValAspLysGlu                               545550555560                                                                   TATCTTTTAGAAAAAATATCTTCATTAGCAAGAAGTTCAGAGAGAGGA1728                           TyrLeuLeuGluLysIleSerSerLeuAlaArgSerSerGluArgGly                               565570575                                                                      TATATACACTATATTGTTCAGTTACAAGGAGATAAAATTAGTTATGAA1776                           TyrIleHisTyrIleValGlnLeuGlnGlyAspLysIleSerTyrGlu                               580585590                                                                      GCAGCATGTAACTTATTTGCAAAGACTCCTTATGATAGTGTACTGTTT1824                           AlaAlaCysAsnLeuPheAlaLysThrProTyrAspSerValLeuPhe                               595600605                                                                      CAGAAAAATATAGAAGATTCAGAAATTGCATATTATTATAATCCTGGA1872                           GlnLysAsnIleGluAspSerGluIleAlaTyrTyrTyrAsnProGly                               610615620                                                                      GATGGTGAAATACAAGAAATAGACAAGTATAAAATTCCAAGTATAATT1920                           AspGlyGluIleGlnGluIleAspLysTyrLysIleProSerIleIle                               625630635640                                                                   TCTGATAGACCTAAGATTAAATTAACATTTATTGGTCATGGTAAAGAT1968                           SerAspArgProLysIleLysLeuThrPheIleGlyHisGlyLysAsp                               645650655                                                                      GAATTTAATACTGATATATTTGCAGGTTTTGATGTAGATTCATTATCC2016                           GluPheAsnThrAspIlePheAlaGlyPheAspValAspSerLeuSer                               660665670                                                                      ACAGAAATAGAAGCAGCAATAGATTTAGCTAAAGAGGATATTTCTCCT2064                           ThrGluIleGluAlaAlaIleAspLeuAlaLysGluAspIleSerPro                               675680685                                                                      AAGTCAATAGAAATAAATTTATTAGGATGTAATATGTTTAGCTACTCT2112                           LysSerIleGluIleAsnLeuLeuGlyCysAsnMetPheSerTyrSer                               690695700                                                                      ATCAACGTAGAGGAGACTTATCCTGGAAAATTATTACTTAAAGTTAAA2160                           IleAsnValGluGluThrTyrProGlyLysLeuLeuLeuLysValLys                               705710715720                                                                   GATAAAATATCAGAATTAATGCCATCTATAAGTCAAGACTCTATTATA2208                           AspLysIleSerGluLeuMetProSerIleSerGlnAspSerIleIle                               725730735                                                                      GTAAGTGCAAATCAATATGAAGTTAGAATAAATAGTGAAGGAAGAAGA2256                           ValSerAlaAsnGlnTyrGluValArgIleAsnSerGluGlyArgArg                               740745750                                                                      GAATTATTGGATCATTCTGGTGAATGGATAAATAAAGAAGAAAGTATT2304                           GluLeuLeuAspHisSerGlyGluTrpIleAsnLysGluGluSerIle                               755760765                                                                      ATAAAGGATATTTCATCAAAAGAATATATATCATTTAATCCTAAAGAA2352                           IleLysAspIleSerSerLysGluTyrIleSerPheAsnProLysGlu                               770775780                                                                      AATAAAATTACAGTAAAATCTAAAAATTTACCTGAGCTATCTACATTA2400                           AsnLysIleThrValLysSerLysAsnLeuProGluLeuSerThrLeu                               785790795800                                                                   TTACAAGAAATTAGAAATAATTCTAATTCAAGTGATATTGAACTAGAA2448                           LeuGlnGluIleArgAsnAsnSerAsnSerSerAspIleGluLeuGlu                               805810815                                                                      GAAAAAGTAATGTTAACAGAATGTGAGATAAATGTTATTTCAAATATA2496                           GluLysValMetLeuThrGluCysGluIleAsnValIleSerAsnIle                               820825830                                                                      GATACGCAAATTGTTGAGGAAAGGATTGAAGAAGCTAAGAATTTAACT2544                           AspThrGlnIleValGluGluArgIleGluGluAlaLysAsnLeuThr                               835840845                                                                      TCTGACTCTATTAATTATATAAAAGATGAATTTAAACTAATAGAATCT2592                           SerAspSerIleAsnTyrIleLysAspGluPheLysLeuIleGluSer                               850855860                                                                      ATTTCTGATGCACTATGTGACTTAAAACAACAGAATGAATTAGAAGAT2640                           IleSerAspAlaLeuCysAspLeuLysGlnGlnAsnGluLeuGluAsp                               865870875880                                                                   TCTCATTTTATATCTTTTGAGGACATATCAGAGACTGATGAGGGATTT2688                           SerHisPheIleSerPheGluAspIleSerGluThrAspGluGlyPhe                               885890895                                                                      AGTATAAGATTTATTAATAAAGAAACTGGAGAATCTATATTTGTAGAA2736                           SerIleArgPheIleAsnLysGluThrGlyGluSerIlePheValGlu                               900905910                                                                      ACTGAAAAAACAATATTCTCTGAATATGCTAATCATATAACTGAAGAG2784                           ThrGluLysThrIlePheSerGluTyrAlaAsnHisIleThrGluGlu                               915920925                                                                      ATTTCTAAGATAAAAGGTACTATATTTGATACTGTAAATGGTAAGTTA2832                           IleSerLysIleLysGlyThrIlePheAspThrValAsnGlyLysLeu                               930935940                                                                      GTAAAAAAAGTAAATTTAGATACTACACACGAAGTAAATACTTTAAAT2880                           ValLysLysValAsnLeuAspThrThrHisGluValAsnThrLeuAsn                               945950955960                                                                   GCTGCATTTTTTATACAATCATTAATAGAATATAATAGTTCTAAAGAA2928                           AlaAlaPhePheIleGlnSerLeuIleGluTyrAsnSerSerLysGlu                               965970975                                                                      TCTCTTAGTAATTTAAGTGTAGCAATGAAAGTCCAAGTTTACGCTCAA2976                           SerLeuSerAsnLeuSerValAlaMetLysValGlnValTyrAlaGln                               980985990                                                                      TTATTTAGTACTGGTTTAAATACTATTACAGATGCAGCCAAAGTTGTT3024                           LeuPheSerThrGlyLeuAsnThrIleThrAspAlaAlaLysValVal                               99510001005                                                                    GAATTAGTATCAACTGCATTAGATGAAACTATAGACTTACTTCCTACA3072                           GluLeuValSerThrAlaLeuAspGluThrIleAspLeuLeuProThr                               101010151020                                                                   TTATCTGAAGGATTACCTATAATTGCAACTATTATAGATGGTGTAAGT3120                           LeuSerGluGlyLeuProIleIleAlaThrIleIleAspGlyValSer                               1025103010351040                                                               TTAGGTGCAGCAATCAAAGAGCTAAGTGAAACGAGTGACCCATTATTA3168                           LeuGlyAlaAlaIleLysGluLeuSerGluThrSerAspProLeuLeu                               104510501055                                                                   AGACAAGAAATAGAAGCTAAGATAGGTATAATGGCAGTAAATTTAACA3216                           ArgGlnGluIleGluAlaLysIleGlyIleMetAlaValAsnLeuThr                               106010651070                                                                   ACAGCTACAACTGCAATCATTACTTCATCTTTGGGGATAGCTAGTGGA3264                           ThrAlaThrThrAlaIleIleThrSerSerLeuGlyIleAlaSerGly                               107510801085                                                                   TTTAGTATACTTTTAGTTCCTTTAGCAGGAATTTCAGCAGGTATACCA3312                           PheSerIleLeuLeuValProLeuAlaGlyIleSerAlaGlyIlePro                               109010951100                                                                   AGCTTAGTAAACAATGAACTTGTACTTCGAGATAAGGCAACAAAGGTT3360                           SerLeuValAsnAsnGluLeuValLeuArgAspLysAlaThrLysVal                               1105111011151120                                                               GTAGATTATTTTAAACATGTTTCATTAGTTGAAACTGAAGGAGTATTT3408                           ValAspTyrPheLysHisValSerLeuValGluThrGluGlyValPhe                               112511301135                                                                   ACTTTATTAGATGATAAAATAATGATGCCACAAGATGATTTAGTGATA3456                           ThrLeuLeuAspAspLysIleMetMetProGlnAspAspLeuValIle                               114011451150                                                                   TCAGAAATAGATTTTAATAATAATTCAATAGTTTTAGGTAAATGTGAA3504                           SerGluIleAspPheAsnAsnAsnSerIleValLeuGlyLysCysGlu                               115511601165                                                                   ATCTGGAGAATGGAAGGTGGTTCAGGTCATACTGTAACTGATGATATA3552                           IleTrpArgMetGluGlyGlySerGlyHisThrValThrAspAspIle                               117011751180                                                                   GATCACTTCTTTTCAGCACCATCAATAACATATAGAGAGCCACACTTA3600                           AspHisPhePheSerAlaProSerIleThrTyrArgGluProHisLeu                               1185119011951200                                                               TCTATATATGACGTATTGGAAGTACAAAAAGAAGAACTTGATTTGTCA3648                           SerIleTyrAspValLeuGluValGlnLysGluGluLeuAspLeuSer                               120512101215                                                                   AAAGATTTAATGGTATTACCTAATGCTCCAAATAGAGTATTTGCTTGG3696                           LysAspLeuMetValLeuProAsnAlaProAsnArgValPheAlaTrp                               122012251230                                                                   GAAACAGGATGGACACCAGGTTTAAGAAGCTTAGAAAATGATGGCACA3744                           GluThrGlyTrpThrProGlyLeuArgSerLeuGluAsnAspGlyThr                               123512401245                                                                   AAACTGTTAGACCGTATAAGAGATAACTATGAAGGTGAGTTTTATTGG3792                           LysLeuLeuAspArgIleArgAspAsnTyrGluGlyGluPheTyrTrp                               125012551260                                                                   AGATATTTTGCTTTTATAGCTGATGCTTTAATAACAACATTAAAACCA3840                           ArgTyrPheAlaPheIleAlaAspAlaLeuIleThrThrLeuLysPro                               1265127012751280                                                               AGATATGAAGATACTAATATAAGAATAAATTTAGATAGTAATACTAGA3888                           ArgTyrGluAspThrAsnIleArgIleAsnLeuAspSerAsnThrArg                               128512901295                                                                   AGTTTTATAGTTCCAATAATAACTACAGAATATATAAGAGAAAAATTA3936                           SerPheIleValProIleIleThrThrGluTyrIleArgGluLysLeu                               130013051310                                                                   TCATATTCTTTCTATGGTTCAGGAGGAACTTATGCATTGTCTCTTTCT3984                           SerTyrSerPheTyrGlySerGlyGlyThrTyrAlaLeuSerLeuSer                               131513201325                                                                   CAATATAATATGGGTATAAATATAGAATTAAGTGAAAGTGATGTTTGG4032                           GlnTyrAsnMetGlyIleAsnIleGluLeuSerGluSerAspValTrp                               133013351340                                                                   ATTATAGATGTTGATAATGTTGTGAGAGATGTAACTATAGAATCTGAT4080                           IleIleAspValAspAsnValValArgAspValThrIleGluSerAsp                               1345135013551360                                                               AAAATTAAAAAAGGTGATTTAATAGAAGGTATTTTATCTACACTAAGT4128                           LysIleLysLysGlyAspLeuIleGluGlyIleLeuSerThrLeuSer                               136513701375                                                                   ATTGAAGAGAATAAAATTATCTTAAATAGCCATGAGATTAATTTTTCT4176                           IleGluGluAsnLysIleIleLeuAsnSerHisGluIleAsnPheSer                               138013851390                                                                   GGTGAGGTAAATGGAAGTAATGGATTTGTTTCTTTAACATTTTCAATT4224                           GlyGluValAsnGlySerAsnGlyPheValSerLeuThrPheSerIle                               139514001405                                                                   TTAGAAGGAATAAATGCAATTATAGAAGTTGATTTATTATCTAAATCA4272                           LeuGluGlyIleAsnAlaIleIleGluValAspLeuLeuSerLysSer                               141014151420                                                                   TATAAATTACTTATTTCTGGCGAATTAAAAATATTGATGTTAAATTCA4320                           TyrLysLeuLeuIleSerGlyGluLeuLysIleLeuMetLeuAsnSer                               1425143014351440                                                               AATCATATTCAACAGAAAATAGATTATATAGGATTCAATAGCGAATTA4368                           AsnHisIleGlnGlnLysIleAspTyrIleGlyPheAsnSerGluLeu                               144514501455                                                                   CAGAAAAATATACCATATAGCTTTGTAGATAGTGAAGGAAAAGAGAAT4416                           GlnLysAsnIleProTyrSerPheValAspSerGluGlyLysGluAsn                               146014651470                                                                   GGTTTTATTAATGGTTCAACAAAAGAAGGTTTATTTGTATCTGAATTA4464                           GlyPheIleAsnGlySerThrLysGluGlyLeuPheValSerGluLeu                               147514801485                                                                   CCTGATGTAGTTCTTATAAGTAAGGTTTATATGGATGATAGTAAGCCT4512                           ProAspValValLeuIleSerLysValTyrMetAspAspSerLysPro                               149014951500                                                                   TCATTTGGATATTATAGTAATAATTTGAAAGATGTCAAAGTTATAACT4560                           SerPheGlyTyrTyrSerAsnAsnLeuLysAspValLysValIleThr                               1505151015151520                                                               AAAGATAATGTTAATATATTAACAGGTTATTATCTTAAGGATGATATA4608                           LysAspAsnValAsnIleLeuThrGlyTyrTyrLeuLysAspAspIle                               152515301535                                                                   AAAATCTCTCTTTCTTTGACTCTACAAGATGAAAAAACTATAAAGTTA4656                           LysIleSerLeuSerLeuThrLeuGlnAspGluLysThrIleLysLeu                               154015451550                                                                   AATAGTGTGCATTTAGATGAAAGTGGAGTAGCTGAGATTTTGAAGTTC4704                           AsnSerValHisLeuAspGluSerGlyValAlaGluIleLeuLysPhe                               155515601565                                                                   ATGAATAGAAAAGGTAATACAAATACTTCAGATTCTTTAATGAGCTTT4752                           MetAsnArgLysGlyAsnThrAsnThrSerAspSerLeuMetSerPhe                               157015751580                                                                   TTAGAAAGTATGAATATAAAAAGTATTTTCGTTAATTTCTTACAATCT4800                           LeuGluSerMetAsnIleLysSerIlePheValAsnPheLeuGlnSer                               1585159015951600                                                               AATATTAAGTTTATATTAGATGCTAATTTTATAATAAGTGGTACTACT4848                           AsnIleLysPheIleLeuAspAlaAsnPheIleIleSerGlyThrThr                               160516101615                                                                   TCTATTGGCCAATTTGAGTTTATTTGTGATGAAAATGATAATATACAA4896                           SerIleGlyGlnPheGluPheIleCysAspGluAsnAspAsnIleGln                               162016251630                                                                   CCATATTTCATTAAGTTTAATACACTAGAAACTAATTATACTTTATAT4944                           ProTyrPheIleLysPheAsnThrLeuGluThrAsnTyrThrLeuTyr                               163516401645                                                                   GTAGGAAATAGACAAAATATGATAGTGGAACCAAATTATGATTTAGAT4992                           ValGlyAsnArgGlnAsnMetIleValGluProAsnTyrAspLeuAsp                               165016551660                                                                   GATTCTGGAGATATATCTTCAACTGTTATCAATTTCTCTCAAAAGTAT5040                           AspSerGlyAspIleSerSerThrValIleAsnPheSerGlnLysTyr                               1665167016751680                                                               CTTTATGGAATAGACAGTTGTGTTAATAAAGTTGTAATTTCACCAAAT5088                           LeuTyrGlyIleAspSerCysValAsnLysValValIleSerProAsn                               168516901695                                                                   ATTTATACAGATGAAATAAATATAACGCCTGTATATGAAACAAATAAT5136                           IleTyrThrAspGluIleAsnIleThrProValTyrGluThrAsnAsn                               170017051710                                                                   ACTTATCCAGAAGTTATTGTATTAGATGCAAATTATATAAATGAAAAA5184                           ThrTyrProGluValIleValLeuAspAlaAsnTyrIleAsnGluLys                               171517201725                                                                   ATAAATGTTAATATCAATGATCTATCTATACGATATGTATGGAGTAAT5232                           IleAsnValAsnIleAsnAspLeuSerIleArgTyrValTrpSerAsn                               173017351740                                                                   GATGGTAATGATTTTATTCTTATGTCAACTAGTGAAGAAAATAAGGTG5280                           AspGlyAsnAspPheIleLeuMetSerThrSerGluGluAsnLysVal                               1745175017551760                                                               TCACAAGTTAAAATAAGATTCGTTAATGTTTTTAAAGATAAGACTTTG5328                           SerGlnValLysIleArgPheValAsnValPheLysAspLysThrLeu                               176517701775                                                                   GCAAATAAGCTATCTTTTAACTTTAGTGATAAACAAGATGTACCTGTA5376                           AlaAsnLysLeuSerPheAsnPheSerAspLysGlnAspValProVal                               178017851790                                                                   AGTGAAATAATCTTATCATTTACACCTTCATATTATGAGGATGGATTG5424                           SerGluIleIleLeuSerPheThrProSerTyrTyrGluAspGlyLeu                               179518001805                                                                   ATTGGCTATGATTTGGGTCTAGTTTCTTTATATAATGAGAAATTTTAT5472                           IleGlyTyrAspLeuGlyLeuValSerLeuTyrAsnGluLysPheTyr                               181018151820                                                                   ATTAATAACTTTGGAATGATGGTATCTGGATTAATATATATTAATGAT5520                           IleAsnAsnPheGlyMetMetValSerGlyLeuIleTyrIleAsnAsp                               1825183018351840                                                               TCATTATATTATTTTAAACCACCAGTAAATAATTTGATAACTGGATTT5568                           SerLeuTyrTyrPheLysProProValAsnAsnLeuIleThrGlyPhe                               184518501855                                                                   GTGACTGTAGGCGATGATAAATACTACTTTAATCCAATTAATGGTGGA5616                           ValThrValGlyAspAspLysTyrTyrPheAsnProIleAsnGlyGly                               186018651870                                                                   GCTGCTTCAATTGGAGAGACAATAATTGATGACAAAAATTATTATTTC5664                           AlaAlaSerIleGlyGluThrIleIleAspAspLysAsnTyrTyrPhe                               187518801885                                                                   AACCAAAGTGGAGTGTTACAAACAGGTGTATTTAGTACAGAAGATGGA5712                           AsnGlnSerGlyValLeuGlnThrGlyValPheSerThrGluAspGly                               189018951900                                                                   TTTAAATATTTTGCCCCAGCTAATACACTTGATGAAAACCTAGAAGGA5760                           PheLysTyrPheAlaProAlaAsnThrLeuAspGluAsnLeuGluGly                               1905191019151920                                                               GAAGCAATTGATTTTACTGGAAAATTAATTATTGACGAAAATATTTAT5808                           GluAlaIleAspPheThrGlyLysLeuIleIleAspGluAsnIleTyr                               192519301935                                                                   TATTTTGATGATAATTATAGAGGAGCTGTAGAATGGAAAGAATTAGAT5856                           TyrPheAspAspAsnTyrArgGlyAlaValGluTrpLysGluLeuAsp                               194019451950                                                                   GGTGAAATGCACTATTTTAGCCCAGAAACAGGTAAAGCTTTTAAAGGT5904                           GlyGluMetHisTyrPheSerProGluThrGlyLysAlaPheLysGly                               195519601965                                                                   CTAAATCAAATAGGTGATTATAAATACTATTTCAATTCTGATGGAGTT5952                           LeuAsnGlnIleGlyAspTyrLysTyrTyrPheAsnSerAspGlyVal                               197019751980                                                                   ATGCAAAAAGGATTTGTTAGTATAAATGATAATAAACACTATTTTGAT6000                           MetGlnLysGlyPheValSerIleAsnAspAsnLysHisTyrPheAsp                               1985199019952000                                                               GATTCTGGTGTTATGAAAGTAGGTTACACTGAAATAGATGGCAAGCAT6048                           AspSerGlyValMetLysValGlyTyrThrGluIleAspGlyLysHis                               200520102015                                                                   TTCTACTTTGCTGAAAACGGAGAAATGCAAATAGGAGTATTTAATACA6096                           PheTyrPheAlaGluAsnGlyGluMetGlnIleGlyValPheAsnThr                               202020252030                                                                   GAAGATGGATTTAAATATTTTGCTCATCATAATGAAGATTTAGGAAAT6144                           GluAspGlyPheLysTyrPheAlaHisHisAsnGluAspLeuGlyAsn                               203520402045                                                                   GAAGAAGGTGAAGAAATCTCATATTCTGGTATATTAAATTTCAATAAT6192                           GluGluGlyGluGluIleSerTyrSerGlyIleLeuAsnPheAsnAsn                               205020552060                                                                   AAAATTTACTATTTTGATGATTCATTTACAGCTGTAGTTGGATGGAAA6240                           LysIleTyrTyrPheAspAspSerPheThrAlaValValGlyTrpLys                               2065207020752080                                                               GATTTAGAGGATGGTTCAAAGTATTATTTTGATGAAGATACAGCAGAA6288                           AspLeuGluAspGlySerLysTyrTyrPheAspGluAspThrAlaGlu                               208520902095                                                                   GCATATATAGGTTTGTCATTAATAAATGATGGTCAATATTATTTTAAT6336                           AlaTyrIleGlyLeuSerLeuIleAsnAspGlyGlnTyrTyrPheAsn                               210021052110                                                                   GATGATGGAATTATGCAAGTTGGATTTGTCACTATAAATGATAAAGTC6384                           AspAspGlyIleMetGlnValGlyPheValThrIleAsnAspLysVal                               211521202125                                                                   TTCTACTTCTCTGACTCTGGAATTATAGAATCTGGAGTACAAAACATA6432                           PheTyrPheSerAspSerGlyIleIleGluSerGlyValGlnAsnIle                               213021352140                                                                   GATGACAATTATTTCTATATAGATGATAATGGTATAGTTCAAATTGGT6480                           AspAspAsnTyrPheTyrIleAspAspAsnGlyIleValGlnIleGly                               2145215021552160                                                               GTATTTGATACTTCAGATGGATATAAATATTTTGCACCTGCTAATACT6528                           ValPheAspThrSerAspGlyTyrLysTyrPheAlaProAlaAsnThr                               216521702175                                                                   GTAAATGATAATATTTACGGACAAGCAGTTGAATATAGTGGTTTAGTT6576                           ValAsnAspAsnIleTyrGlyGlnAlaValGluTyrSerGlyLeuVal                               218021852190                                                                   AGAGTTGGGGAAGATGTATATTATTTTGGAGAAACATATACAATTGAG6624                           ArgValGlyGluAspValTyrTyrPheGlyGluThrTyrThrIleGlu                               219522002205                                                                   ACTGGATGGATATATGATATGGAAAATGAAAGTGATAAATATTATTTC6672                           ThrGlyTrpIleTyrAspMetGluAsnGluSerAspLysTyrTyrPhe                               221022152220                                                                   AATCCAGAAACTAAAAAAGCATGCAAAGGTATTAATTTAATTGATGAT6720                           AsnProGluThrLysLysAlaCysLysGlyIleAsnLeuIleAspAsp                               2225223022352240                                                               ATAAAATATTATTTTGATGAGAAGGGCATAATGAGAACGGGTCTTATA6768                           IleLysTyrTyrPheAspGluLysGlyIleMetArgThrGlyLeuIle                               224522502255                                                                   TCATTTGAAAATAATAATTATTACTTTAATGAGAATGGTGAAATGCAA6816                           SerPheGluAsnAsnAsnTyrTyrPheAsnGluAsnGlyGluMetGln                               226022652270                                                                   TTTGGTTATATAAATATAGAAGATAAGATGTTCTATTTTGGTGAAGAT6864                           PheGlyTyrIleAsnIleGluAspLysMetPheTyrPheGlyGluAsp                               227522802285                                                                   GGTGTCATGCAGATTGGAGTATTTAATACACCAGATGGATTTAAATAC6912                           GlyValMetGlnIleGlyValPheAsnThrProAspGlyPheLysTyr                               229022952300                                                                   TTTGCACATCAAAATACTTTGGATGAGAATTTTGAGGGAGAATCAATA6960                           PheAlaHisGlnAsnThrLeuAspGluAsnPheGluGlyGluSerIle                               2305231023152320                                                               AACTATACTGGTTGGTTAGATTTAGATGAAAAGAGATATTATTTTACA7008                           AsnTyrThrGlyTrpLeuAspLeuAspGluLysArgTyrTyrPheThr                               232523302335                                                                   GATGAATATATTGCAGCAACTGGTTCAGTTATTATTGATGGTGAGGAG7056                           AspGluTyrIleAlaAlaThrGlySerValIleIleAspGlyGluGlu                               234023452350                                                                   TATTATTTTGATCCTGATACAGCTCAATTAGTGATTAGTGAA7098                                 TyrTyrPheAspProAspThrAlaGlnLeuValIleSerGlu                                     235523602365                                                                   TAG7101                                                                        (2) INFORMATION FOR SEQ ID NO:10:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2366 amino acids                                                   (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                       MetSerLeuValAsnArgLysGlnLeuGluLysMetAlaAsnValArg                               151015                                                                         PheArgThrGlnGluAspGluTyrValAlaIleLeuAspAlaLeuGlu                               202530                                                                         GluTyrHisAsnMetSerGluAsnThrValValGluLysTyrLeuLys                               354045                                                                         LeuLysAspIleAsnSerLeuThrAspIleTyrIleAspThrTyrLys                               505560                                                                         LysSerGlyArgAsnLysAlaLeuLysLysPheLysGluTyrLeuVal                               65707580                                                                       ThrGluValLeuGluLeuLysAsnAsnAsnLeuThrProValGluLys                               859095                                                                         AsnLeuHisPheValTrpIleGlyGlyGlnIleAsnAspThrAlaIle                               100105110                                                                      AsnTyrIleAsnGlnTrpLysAspValAsnSerAspTyrAsnValAsn                               115120125                                                                      ValPheTyrAspSerAsnAlaPheLeuIleAsnThrLeuLysLysThr                               130135140                                                                      ValValGluSerAlaIleAsnAspThrLeuGluSerPheArgGluAsn                               145150155160                                                                   LeuAsnAspProArgPheAspTyrAsnLysPhePheArgLysArgMet                               165170175                                                                      GluIleIleTyrAspLysGlnLysAsnPheIleAsnTyrTyrLysAla                               180185190                                                                      GlnArgGluGluAsnProGluLeuIleIleAspAspIleValLysThr                               195200205                                                                      TyrLeuSerAsnGluTyrSerLysGluIleAspGluLeuAsnThrTyr                               210215220                                                                      IleGluGluSerLeuAsnLysIleThrGlnAsnSerGlyAsnAspVal                               225230235240                                                                   ArgAsnPheGluGluPheLysAsnGlyGluSerPheAsnLeuTyrGlu                               245250255                                                                      GlnGluLeuValGluArgTrpAsnLeuAlaAlaAlaSerAspIleLeu                               260265270                                                                      ArgIleSerAlaLeuLysGluIleGlyGlyMetTyrLeuAspValAsp                               275280285                                                                      MetLeuProGlyIleGlnProAspLeuPheGluSerIleGluLysPro                               290295300                                                                      SerSerValThrValAspPheTrpGluMetThrLysLeuGluAlaIle                               305310315320                                                                   MetLysTyrLysGluTyrIleProGluTyrThrSerGluHisPheAsp                               325330335                                                                      MetLeuAspGluGluValGlnSerSerPheGluSerValLeuAlaSer                               340345350                                                                      LysSerAspLysSerGluIlePheSerSerLeuGlyAspMetGluAla                               355360365                                                                      SerProLeuGluValLysIleAlaPheAsnSerLysGlyIleIleAsn                               370375380                                                                      GlnGlyLeuIleSerValLysAspSerTyrCysSerAsnLeuIleVal                               385390395400                                                                   LysGlnIleGluAsnArgTyrLysIleLeuAsnAsnSerLeuAsnPro                               405410415                                                                      AlaIleSerGluAspAsnAspPheAsnThrThrThrAsnThrPheIle                               420425430                                                                      AspSerIleMetAlaGluAlaAsnAlaAspAsnGlyArgPheMetMet                               435440445                                                                      GluLeuGlyLysTyrLeuArgValGlyPhePheProAspValLysThr                               450455460                                                                      ThrIleAsnLeuSerGlyProGluAlaTyrAlaAlaAlaTyrGlnAsp                               465470475480                                                                   LeuLeuMetPheLysGluGlySerMetAsnIleHisLeuIleGluAla                               485490495                                                                      AspLeuArgAsnPheGluIleSerLysThrAsnIleSerGlnSerThr                               500505510                                                                      GluGlnGluMetAlaSerLeuTrpSerPheAspAspAlaArgAlaLys                               515520525                                                                      AlaGlnPheGluGluTyrLysArgAsnTyrPheGluGlySerLeuGly                               530535540                                                                      GluAspAspAsnLeuAspPheSerGlnAsnIleValValAspLysGlu                               545550555560                                                                   TyrLeuLeuGluLysIleSerSerLeuAlaArgSerSerGluArgGly                               565570575                                                                      TyrIleHisTyrIleValGlnLeuGlnGlyAspLysIleSerTyrGlu                               580585590                                                                      AlaAlaCysAsnLeuPheAlaLysThrProTyrAspSerValLeuPhe                               595600605                                                                      GlnLysAsnIleGluAspSerGluIleAlaTyrTyrTyrAsnProGly                               610615620                                                                      AspGlyGluIleGlnGluIleAspLysTyrLysIleProSerIleIle                               625630635640                                                                   SerAspArgProLysIleLysLeuThrPheIleGlyHisGlyLysAsp                               645650655                                                                      GluPheAsnThrAspIlePheAlaGlyPheAspValAspSerLeuSer                               660665670                                                                      ThrGluIleGluAlaAlaIleAspLeuAlaLysGluAspIleSerPro                               675680685                                                                      LysSerIleGluIleAsnLeuLeuGlyCysAsnMetPheSerTyrSer                               690695700                                                                      IleAsnValGluGluThrTyrProGlyLysLeuLeuLeuLysValLys                               705710715720                                                                   AspLysIleSerGluLeuMetProSerIleSerGlnAspSerIleIle                               725730735                                                                      ValSerAlaAsnGlnTyrGluValArgIleAsnSerGluGlyArgArg                               740745750                                                                      GluLeuLeuAspHisSerGlyGluTrpIleAsnLysGluGluSerIle                               755760765                                                                      IleLysAspIleSerSerLysGluTyrIleSerPheAsnProLysGlu                               770775780                                                                      AsnLysIleThrValLysSerLysAsnLeuProGluLeuSerThrLeu                               785790795800                                                                   LeuGlnGluIleArgAsnAsnSerAsnSerSerAspIleGluLeuGlu                               805810815                                                                      GluLysValMetLeuThrGluCysGluIleAsnValIleSerAsnIle                               820825830                                                                      AspThrGlnIleValGluGluArgIleGluGluAlaLysAsnLeuThr                               835840845                                                                      SerAspSerIleAsnTyrIleLysAspGluPheLysLeuIleGluSer                               850855860                                                                      IleSerAspAlaLeuCysAspLeuLysGlnGlnAsnGluLeuGluAsp                               865870875880                                                                   SerHisPheIleSerPheGluAspIleSerGluThrAspGluGlyPhe                               885890895                                                                      SerIleArgPheIleAsnLysGluThrGlyGluSerIlePheValGlu                               900905910                                                                      ThrGluLysThrIlePheSerGluTyrAlaAsnHisIleThrGluGlu                               915920925                                                                      IleSerLysIleLysGlyThrIlePheAspThrValAsnGlyLysLeu                               930935940                                                                      ValLysLysValAsnLeuAspThrThrHisGluValAsnThrLeuAsn                               945950955960                                                                   AlaAlaPhePheIleGlnSerLeuIleGluTyrAsnSerSerLysGlu                               965970975                                                                      SerLeuSerAsnLeuSerValAlaMetLysValGlnValTyrAlaGln                               980985990                                                                      LeuPheSerThrGlyLeuAsnThrIleThrAspAlaAlaLysValVal                               99510001005                                                                    GluLeuValSerThrAlaLeuAspGluThrIleAspLeuLeuProThr                               101010151020                                                                   LeuSerGluGlyLeuProIleIleAlaThrIleIleAspGlyValSer                               1025103010351040                                                               LeuGlyAlaAlaIleLysGluLeuSerGluThrSerAspProLeuLeu                               104510501055                                                                   ArgGlnGluIleGluAlaLysIleGlyIleMetAlaValAsnLeuThr                               106010651070                                                                   ThrAlaThrThrAlaIleIleThrSerSerLeuGlyIleAlaSerGly                               107510801085                                                                   PheSerIleLeuLeuValProLeuAlaGlyIleSerAlaGlyIlePro                               109010951100                                                                   SerLeuValAsnAsnGluLeuValLeuArgAspLysAlaThrLysVal                               1105111011151120                                                               ValAspTyrPheLysHisValSerLeuValGluThrGluGlyValPhe                               112511301135                                                                   ThrLeuLeuAspAspLysIleMetMetProGlnAspAspLeuValIle                               114011451150                                                                   SerGluIleAspPheAsnAsnAsnSerIleValLeuGlyLysCysGlu                               115511601165                                                                   IleTrpArgMetGluGlyGlySerGlyHisThrValThrAspAspIle                               117011751180                                                                   AspHisPhePheSerAlaProSerIleThrTyrArgGluProHisLeu                               1185119011951200                                                               SerIleTyrAspValLeuGluValGlnLysGluGluLeuAspLeuSer                               120512101215                                                                   LysAspLeuMetValLeuProAsnAlaProAsnArgValPheAlaTrp                               122012251230                                                                   GluThrGlyTrpThrProGlyLeuArgSerLeuGluAsnAspGlyThr                               123512401245                                                                   LysLeuLeuAspArgIleArgAspAsnTyrGluGlyGluPheTyrTrp                               125012551260                                                                   ArgTyrPheAlaPheIleAlaAspAlaLeuIleThrThrLeuLysPro                               1265127012751280                                                               ArgTyrGluAspThrAsnIleArgIleAsnLeuAspSerAsnThrArg                               128512901295                                                                   SerPheIleValProIleIleThrThrGluTyrIleArgGluLysLeu                               130013051310                                                                   SerTyrSerPheTyrGlySerGlyGlyThrTyrAlaLeuSerLeuSer                               131513201325                                                                   GlnTyrAsnMetGlyIleAsnIleGluLeuSerGluSerAspValTrp                               133013351340                                                                   IleIleAspValAspAsnValValArgAspValThrIleGluSerAsp                               1345135013551360                                                               LysIleLysLysGlyAspLeuIleGluGlyIleLeuSerThrLeuSer                               136513701375                                                                   IleGluGluAsnLysIleIleLeuAsnSerHisGluIleAsnPheSer                               138013851390                                                                   GlyGluValAsnGlySerAsnGlyPheValSerLeuThrPheSerIle                               139514001405                                                                   LeuGluGlyIleAsnAlaIleIleGluValAspLeuLeuSerLysSer                               141014151420                                                                   TyrLysLeuLeuIleSerGlyGluLeuLysIleLeuMetLeuAsnSer                               1425143014351440                                                               AsnHisIleGlnGlnLysIleAspTyrIleGlyPheAsnSerGluLeu                               144514501455                                                                   GlnLysAsnIleProTyrSerPheValAspSerGluGlyLysGluAsn                               146014651470                                                                   GlyPheIleAsnGlySerThrLysGluGlyLeuPheValSerGluLeu                               147514801485                                                                   ProAspValValLeuIleSerLysValTyrMetAspAspSerLysPro                               149014951500                                                                   SerPheGlyTyrTyrSerAsnAsnLeuLysAspValLysValIleThr                               1505151015151520                                                               LysAspAsnValAsnIleLeuThrGlyTyrTyrLeuLysAspAspIle                               152515301535                                                                   LysIleSerLeuSerLeuThrLeuGlnAspGluLysThrIleLysLeu                               154015451550                                                                   AsnSerValHisLeuAspGluSerGlyValAlaGluIleLeuLysPhe                               155515601565                                                                   MetAsnArgLysGlyAsnThrAsnThrSerAspSerLeuMetSerPhe                               157015751580                                                                   LeuGluSerMetAsnIleLysSerIlePheValAsnPheLeuGlnSer                               1585159015951600                                                               AsnIleLysPheIleLeuAspAlaAsnPheIleIleSerGlyThrThr                               160516101615                                                                   SerIleGlyGlnPheGluPheIleCysAspGluAsnAspAsnIleGln                               162016251630                                                                   ProTyrPheIleLysPheAsnThrLeuGluThrAsnTyrThrLeuTyr                               163516401645                                                                   ValGlyAsnArgGlnAsnMetIleValGluProAsnTyrAspLeuAsp                               165016551660                                                                   AspSerGlyAspIleSerSerThrValIleAsnPheSerGlnLysTyr                               1665167016751680                                                               LeuTyrGlyIleAspSerCysValAsnLysValValIleSerProAsn                               168516901695                                                                   IleTyrThrAspGluIleAsnIleThrProValTyrGluThrAsnAsn                               170017051710                                                                   ThrTyrProGluValIleValLeuAspAlaAsnTyrIleAsnGluLys                               171517201725                                                                   IleAsnValAsnIleAsnAspLeuSerIleArgTyrValTrpSerAsn                               173017351740                                                                   AspGlyAsnAspPheIleLeuMetSerThrSerGluGluAsnLysVal                               1745175017551760                                                               SerGlnValLysIleArgPheValAsnValPheLysAspLysThrLeu                               176517701775                                                                   AlaAsnLysLeuSerPheAsnPheSerAspLysGlnAspValProVal                               178017851790                                                                   SerGluIleIleLeuSerPheThrProSerTyrTyrGluAspGlyLeu                               179518001805                                                                   IleGlyTyrAspLeuGlyLeuValSerLeuTyrAsnGluLysPheTyr                               181018151820                                                                   IleAsnAsnPheGlyMetMetValSerGlyLeuIleTyrIleAsnAsp                               1825183018351840                                                               SerLeuTyrTyrPheLysProProValAsnAsnLeuIleThrGlyPhe                               184518501855                                                                   ValThrValGlyAspAspLysTyrTyrPheAsnProIleAsnGlyGly                               186018651870                                                                   AlaAlaSerIleGlyGluThrIleIleAspAspLysAsnTyrTyrPhe                               187518801885                                                                   AsnGlnSerGlyValLeuGlnThrGlyValPheSerThrGluAspGly                               189018951900                                                                   PheLysTyrPheAlaProAlaAsnThrLeuAspGluAsnLeuGluGly                               1905191019151920                                                               GluAlaIleAspPheThrGlyLysLeuIleIleAspGluAsnIleTyr                               192519301935                                                                   TyrPheAspAspAsnTyrArgGlyAlaValGluTrpLysGluLeuAsp                               194019451950                                                                   GlyGluMetHisTyrPheSerProGluThrGlyLysAlaPheLysGly                               195519601965                                                                   LeuAsnGlnIleGlyAspTyrLysTyrTyrPheAsnSerAspGlyVal                               197019751980                                                                   MetGlnLysGlyPheValSerIleAsnAspAsnLysHisTyrPheAsp                               1985199019952000                                                               AspSerGlyValMetLysValGlyTyrThrGluIleAspGlyLysHis                               200520102015                                                                   PheTyrPheAlaGluAsnGlyGluMetGlnIleGlyValPheAsnThr                               202020252030                                                                   GluAspGlyPheLysTyrPheAlaHisHisAsnGluAspLeuGlyAsn                               203520402045                                                                   GluGluGlyGluGluIleSerTyrSerGlyIleLeuAsnPheAsnAsn                               205020552060                                                                   LysIleTyrTyrPheAspAspSerPheThrAlaValValGlyTrpLys                               2065207020752080                                                               AspLeuGluAspGlySerLysTyrTyrPheAspGluAspThrAlaGlu                               208520902095                                                                   AlaTyrIleGlyLeuSerLeuIleAsnAspGlyGlnTyrTyrPheAsn                               210021052110                                                                   AspAspGlyIleMetGlnValGlyPheValThrIleAsnAspLysVal                               211521202125                                                                   PheTyrPheSerAspSerGlyIleIleGluSerGlyValGlnAsnIle                               213021352140                                                                   AspAspAsnTyrPheTyrIleAspAspAsnGlyIleValGlnIleGly                               2145215021552160                                                               ValPheAspThrSerAspGlyTyrLysTyrPheAlaProAlaAsnThr                               216521702175                                                                   ValAsnAspAsnIleTyrGlyGlnAlaValGluTyrSerGlyLeuVal                               218021852190                                                                   ArgValGlyGluAspValTyrTyrPheGlyGluThrTyrThrIleGlu                               219522002205                                                                   ThrGlyTrpIleTyrAspMetGluAsnGluSerAspLysTyrTyrPhe                               221022152220                                                                   AsnProGluThrLysLysAlaCysLysGlyIleAsnLeuIleAspAsp                               2225223022352240                                                               IleLysTyrTyrPheAspGluLysGlyIleMetArgThrGlyLeuIle                               224522502255                                                                   SerPheGluAsnAsnAsnTyrTyrPheAsnGluAsnGlyGluMetGln                               226022652270                                                                   PheGlyTyrIleAsnIleGluAspLysMetPheTyrPheGlyGluAsp                               227522802285                                                                   GlyValMetGlnIleGlyValPheAsnThrProAspGlyPheLysTyr                               229022952300                                                                   PheAlaHisGlnAsnThrLeuAspGluAsnPheGluGlyGluSerIle                               2305231023152320                                                               AsnTyrThrGlyTrpLeuAspLeuAspGluLysArgTyrTyrPheThr                               232523302335                                                                   AspGluTyrIleAlaAlaThrGlySerValIleIleAspGlyGluGlu                               234023452350                                                                   TyrTyrPheAspProAspThrAlaGlnLeuValIleSerGlu                                     235523602365                                                                   (2) INFORMATION FOR SEQ ID NO:11:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 19 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                       TAGAAAAAATGGCAAATGT19                                                          (2) INFORMATION FOR SEQ ID NO:12:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                       TTTCATCTTGTAGAGTCAAAG21                                                        (2) INFORMATION FOR SEQ ID NO:13:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 22 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                       GATGCCACAAGATGATTTAGTG22                                                       (2) INFORMATION FOR SEQ ID NO:14:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 22 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                       CTAATTGAGCTGTATCAGGATC22                                                       (2) INFORMATION FOR SEQ ID NO:15:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 27 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                       CGGAATTCCTAGAAAAAATGGCAAATG27                                                  (2) INFORMATION FOR SEQ ID NO:16:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 26 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                       GCTCTAGAATGACCATAAGCTAGCCA26                                                   (2) INFORMATION FOR SEQ ID NO:17:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 27 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                       CGGAATTCGAGTTGGTAGAAAGGTGGA27                                                  (2) INFORMATION FOR SEQ ID NO:18:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 27 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                       CGGAATTCGGTTATTATCTTAAGGATG27                                                  (2) INFORMATION FOR SEQ ID NO:19:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 28 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                       CGGAATTCTTGATAACTGGATTTGTGAC28                                                 (2) INFORMATION FOR SEQ ID NO:20:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 511 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: unknown                                                      (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                       LeuIleThrGlyPheValThrValGlyAspAspLysTyrTyrPheAsn                               151015                                                                         ProIleAsnGlyGlyAlaAlaSerIleGlyGluThrIleIleAspAsp                               202530                                                                         LysAsnTyrTyrPheAsnGlnSerGlyValLeuGlnThrGlyValPhe                               354045                                                                         SerThrGluAspGlyPheLysTyrPheAlaProAlaAsnThrLeuAsp                               505560                                                                         GluAsnLeuGluGlyGluAlaIleAspPheThrGlyLysLeuIleIle                               65707580                                                                       AspGluAsnIleTyrTyrPheAspAspAsnTyrArgGlyAlaValGlu                               859095                                                                         TrpLysGluLeuAspGlyGluMetHisTyrPheSerProGluThrGly                               100105110                                                                      LysAlaPheLysGlyLeuAsnGlnIleGlyAspTyrLysTyrTyrPhe                               115120125                                                                      AsnSerAspGlyValMetGlnLysGlyPheValSerIleAsnAspAsn                               130135140                                                                      LysHisTyrPheAspAspSerGlyValMetLysValGlyTyrThrGlu                               145150155160                                                                   IleAspGlyLysHisPheTyrPheAlaGluAsnGlyGluMetGlnIle                               165170175                                                                      GlyValPheAsnThrGluAspGlyPheLysTyrPheAlaHisHisAsn                               180185190                                                                      GluAspLeuGlyAsnGluGluGlyGluGluIleSerTyrSerGlyIle                               195200205                                                                      LeuAsnPheAsnAsnLysIleTyrTyrPheAspAspSerPheThrAla                               210215220                                                                      ValValGlyTrpLysAspLeuGluAspGlySerLysTyrTyrPheAsp                               225230235240                                                                   GluAspThrAlaGluAlaTyrIleGlyLeuSerLeuIleAsnAspGly                               245250255                                                                      GlnTyrTyrPheAsnAspAspGlyIleMetGlnValGlyPheValThr                               260265270                                                                      IleAsnAspLysValPheTyrPheSerAspSerGlyIleIleGluSer                               275280285                                                                      GlyValGlnAsnIleAspAspAsnTyrPheTyrIleAspAspAsnGly                               290295300                                                                      IleValGlnIleGlyValPheAspThrSerAspGlyTyrLysTyrPhe                               305310315320                                                                   AlaProAlaAsnThrValAsnAspAsnIleTyrGlyGlnAlaValGlu                               325330335                                                                      TyrSerGlyLeuValArgValGlyGluAspValTyrTyrPheGlyGlu                               340345350                                                                      ThrTyrThrIleGluThrGlyTrpIleTyrAspMetGluAsnGluSer                               355360365                                                                      AspLysTyrTyrPheAsnProGluThrLysLysAlaCysLysGlyIle                               370375380                                                                      AsnLeuIleAspAspIleLysTyrTyrPheAspGluLysGlyIleMet                               385390395400                                                                   ArgThrGlyLeuIleSerPheGluAsnAsnAsnTyrTyrPheAsnGlu                               405410415                                                                      AsnGlyGluMetGlnPheGlyTyrIleAsnIleGluAspLysMetPhe                               420425430                                                                      TyrPheGlyGluAspGlyValMetGlnIleGlyValPheAsnThrPro                               435440445                                                                      AspGlyPheLysTyrPheAlaHisGlnAsnThrLeuAspGluAsnPhe                               450455460                                                                      GluGlyGluSerIleAsnTyrThrGlyTrpLeuAspLeuAspGluLys                               465470475480                                                                   ArgTyrTyrPheThrAspGluTyrIleAlaAlaThrGlySerValIle                               485490495                                                                      IleAspGlyGluGluTyrTyrPheAspProAspThrAlaGlnLeu                                  500505510                                                                      (2) INFORMATION FOR SEQ ID NO:21:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 608 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: unknown                                                      (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                       SerGluGluAsnLysValSerGlnValLysIleArgPheValAsnVal                               151015                                                                         PheLysAspLysThrLeuAlaAsnLysLeuSerPheAsnPheSerAsp                               202530                                                                         LysGlnAspValProValSerGluIleIleLeuSerPheThrProSer                               354045                                                                         TyrTyrGluAspGlyLeuIleGlyTyrAspLeuGlyLeuValSerLeu                               505560                                                                         TyrAsnGluLysPheTyrIleAsnAsnPheGlyMetMetValSerGly                               65707580                                                                       LeuIleTyrIleAsnAspSerLeuTyrTyrPheLysProProValAsn                               859095                                                                         AsnLeuIleThrGlyPheValThrValGlyAspAspLysTyrTyrPhe                               100105110                                                                      AsnProIleAsnGlyGlyAlaAlaSerIleGlyGluThrIleIleAsp                               115120125                                                                      AspLysAsnTyrTyrPheAsnGlnSerGlyValLeuGlnThrGlyVal                               130135140                                                                      PheSerThrGluAspGlyPheLysTyrPheAlaProAlaAsnThrLeu                               145150155160                                                                   AspGluAsnLeuGluGlyGluAlaIleAspPheThrGlyLysLeuIle                               165170175                                                                      IleAspGluAsnIleTyrTyrPheAspAspAsnTyrArgGlyAlaVal                               180185190                                                                      GluTrpLysGluLeuAspGlyGluMetHisTyrPheSerProGluThr                               195200205                                                                      GlyLysAlaPheLysGlyLeuAsnGlnIleGlyAspTyrLysTyrTyr                               210215220                                                                      PheAsnSerAspGlyValMetGlnLysGlyPheValSerIleAsnAsp                               225230235240                                                                   AsnLysHisTyrPheAspAspSerGlyValMetLysValGlyTyrThr                               245250255                                                                      GluIleAspGlyLysHisPheTyrPheAlaGluAsnGlyGluMetGln                               260265270                                                                      IleGlyValPheAsnThrGluAspGlyPheLysTyrPheAlaHisHis                               275280285                                                                      AsnGluAspLeuGlyAsnGluGluGlyGluGluIleSerTyrSerGly                               290295300                                                                      IleLeuAsnPheAsnAsnLysIleTyrTyrPheAspAspSerPheThr                               305310315320                                                                   AlaValValGlyTrpLysAspLeuGluAspGlySerLysTyrTyrPhe                               325330335                                                                      AspGluAspThrAlaGluAlaTyrIleGlyLeuSerLeuIleAsnAsp                               340345350                                                                      GlyGlnTyrTyrPheAsnAspAspGlyIleMetGlnValGlyPheVal                               355360365                                                                      ThrIleAsnAspLysValPheTyrPheSerAspSerGlyIleIleGlu                               370375380                                                                      SerGlyValGlnAsnIleAspAspAsnTyrPheTyrIleAspAspAsn                               385390395400                                                                   GlyIleValGlnIleGlyValPheAspThrSerAspGlyTyrLysTyr                               405410415                                                                      PheAlaProAlaAsnThrValAsnAspAsnIleTyrGlyGlnAlaVal                               420425430                                                                      GluTyrSerGlyLeuValArgValGlyGluAspValTyrTyrPheGly                               435440445                                                                      GluThrTyrThrIleGluThrGlyTrpIleTyrAspMetGluAsnGlu                               450455460                                                                      SerAspLysTyrTyrPheAsnProGluThrLysLysAlaCysLysGly                               465470475480                                                                   IleAsnLeuIleAspAspIleLysTyrTyrPheAspGluLysGlyIle                               485490495                                                                      MetArgThrGlyLeuIleSerPheGluAsnAsnAsnTyrTyrPheAsn                               500505510                                                                      GluAsnGlyGluMetGlnPheGlyTyrIleAsnIleGluAspLysMet                               515520525                                                                      PheTyrPheGlyGluAspGlyValMetGlnIleGlyValPheAsnThr                               530535540                                                                      ProAspGlyPheLysTyrPheAlaHisGlnAsnThrLeuAspGluAsn                               545550555560                                                                   PheGluGlyGluSerIleAsnTyrThrGlyTrpLeuAspLeuAspGlu                               565570575                                                                      LysArgTyrTyrPheThrAspGluTyrIleAlaAlaThrGlySerVal                               580585590                                                                      IleIleAspGlyGluGluTyrTyrPheAspProAspThrAlaGlnLeu                               595600605                                                                      (2) INFORMATION FOR SEQ ID NO:22:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1330 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..1314                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                       ATGGCTCGTCTGCTGTCTACCTTCACTGAATACATCAAGAACATCATC48                             MetAlaArgLeuLeuSerThrPheThrGluTyrIleLysAsnIleIle                               151015                                                                         AATACCTCCATCCTGAACCTGCGCTACGAATCCAATCACCTGATCGAC96                             AsnThrSerIleLeuAsnLeuArgTyrGluSerAsnHisLeuIleAsp                               202530                                                                         CTGTCTCGCTACGCTTCCAAAATCAACATCGGTTCTAAAGTTAACTTC144                            LeuSerArgTyrAlaSerLysIleAsnIleGlySerLysValAsnPhe                               354045                                                                         GATCCGATCGACAAGAATCAGATCCAGCTGTTCAATCTGGAATCTTCC192                            AspProIleAspLysAsnGlnIleGlnLeuPheAsnLeuGluSerSer                               505560                                                                         AAAATCGAAGTTATCCTGAAGAATGCTATCGTATACAACTCTATGTAC240                            LysIleGluValIleLeuLysAsnAlaIleValTyrAsnSerMetTyr                               65707580                                                                       GAAAACTTCTCCACCTCCTTCTGGATCCGTATCCCGAAATACTTCAAC288                            GluAsnPheSerThrSerPheTrpIleArgIleProLysTyrPheAsn                               859095                                                                         TCCATCTCTCTGAACAATGAATACACCATCATCAACTGCATGGAAAAC336                            SerIleSerLeuAsnAsnGluTyrThrIleIleAsnCysMetGluAsn                               100105110                                                                      AATTCTGGTTGGAAAGTATCTCTGAACTACGGTGAAATCATCTGGACT384                            AsnSerGlyTrpLysValSerLeuAsnTyrGlyGluIleIleTrpThr                               115120125                                                                      CTGCAGGACACTCAGGAAATCAAACAGCGTGTTGTATTCAAATACTCT432                            LeuGlnAspThrGlnGluIleLysGlnArgValValPheLysTyrSer                               130135140                                                                      CAGATGATCAACATCTCTGACTACATCAATCGCTGGATCTTCGTTACC480                            GlnMetIleAsnIleSerAspTyrIleAsnArgTrpIlePheValThr                               145150155160                                                                   ATCACCAACAATCGTCTGAATAACTCCAAAATCTACATCAACGGCCGT528                            IleThrAsnAsnArgLeuAsnAsnSerLysIleTyrIleAsnGlyArg                               165170175                                                                      CTGATCGACCAGAAACCGATCTCCAATCTGGGTAACATCCACGCTTCT576                            LeuIleAspGlnLysProIleSerAsnLeuGlyAsnIleHisAlaSer                               180185190                                                                      AATAACATCATGTTCAAACTGGACGGTTGTCGTGACACTCACCGCTAC624                            AsnAsnIleMetPheLysLeuAspGlyCysArgAspThrHisArgTyr                               195200205                                                                      ATCTGGATCAAATACTTCAATCTGTTCGACAAAGAACTGAACGAAAAA672                            IleTrpIleLysTyrPheAsnLeuPheAspLysGluLeuAsnGluLys                               210215220                                                                      GAAATCAAAGACCTGTACGACAACCAGTCCAATTCTGGTATCCTGAAA720                            GluIleLysAspLeuTyrAspAsnGlnSerAsnSerGlyIleLeuLys                               225230235240                                                                   GACTTCTGGGGTGACTACCTGCAGTACGACAAACCGTACTACATGCTG768                            AspPheTrpGlyAspTyrLeuGlnTyrAspLysProTyrTyrMetLeu                               245250255                                                                      AATCTGTACGATCCGAACAAATACGTTGACGTCAACAATGTAGGTATC816                            AsnLeuTyrAspProAsnLysTyrValAspValAsnAsnValGlyIle                               260265270                                                                      CGCGGTTACATGTACCTGAAAGGTCCGCGTGGTTCTGTTATGACTACC864                            ArgGlyTyrMetTyrLeuLysGlyProArgGlySerValMetThrThr                               275280285                                                                      AACATCTACCTGAACTCTTCCCTGTACCGTGGTACCAAATTCATCATC912                            AsnIleTyrLeuAsnSerSerLeuTyrArgGlyThrLysPheIleIle                               290295300                                                                      AAGAAATACGCGTCTGGTAACAAGGACAATATCGTTCGCAACAATGAT960                            LysLysTyrAlaSerGlyAsnLysAspAsnIleValArgAsnAsnAsp                               305310315320                                                                   CGTGTATACATCAATGTTGTAGTTAAGAACAAAGAATACCGTCTGGCT1008                           ArgValTyrIleAsnValValValLysAsnLysGluTyrArgLeuAla                               325330335                                                                      ACCAATGCTTCTCAGGCTGGTGTAGAAAAGATCTTGTCTGCTCTGGAA1056                           ThrAsnAlaSerGlnAlaGlyValGluLysIleLeuSerAlaLeuGlu                               340345350                                                                      ATCCCGGACGTTGGTAATCTGTCTCAGGTAGTTGTAATGAAATCCAAG1104                           IleProAspValGlyAsnLeuSerGlnValValValMetLysSerLys                               355360365                                                                      AACGACCAGGGTATCACTAACAAATGCAAAATGAATCTGCAGGACAAC1152                           AsnAspGlnGlyIleThrAsnLysCysLysMetAsnLeuGlnAspAsn                               370375380                                                                      AATGGTAACGATATCGGTTTCATCGGTTTCCACCAGTTCAACAATATC1200                           AsnGlyAsnAspIleGlyPheIleGlyPheHisGlnPheAsnAsnIle                               385390395400                                                                   GCTAAACTGGTTGCTTCCAACTGGTACAATCGTCAGATCGAACGTTCC1248                           AlaLysLeuValAlaSerAsnTrpTyrAsnArgGlnIleGluArgSer                               405410415                                                                      TCTCGCACTCTGGGTTGCTCTTGGGAGTTCATCCCGGTTGATGACGGT1296                           SerArgThrLeuGlyCysSerTrpGluPheIleProValAspAspGly                               420425430                                                                      TGGGGTGAACGTCCGCTGTAACCCGGGAAAGCTT1330                                         TrpGlyGluArgProLeu                                                             435                                                                            (2) INFORMATION FOR SEQ ID NO:23:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 438 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                       MetAlaArgLeuLeuSerThrPheThrGluTyrIleLysAsnIleIle                               151015                                                                         AsnThrSerIleLeuAsnLeuArgTyrGluSerAsnHisLeuIleAsp                               202530                                                                         LeuSerArgTyrAlaSerLysIleAsnIleGlySerLysValAsnPhe                               354045                                                                         AspProIleAspLysAsnGlnIleGlnLeuPheAsnLeuGluSerSer                               505560                                                                         LysIleGluValIleLeuLysAsnAlaIleValTyrAsnSerMetTyr                               65707580                                                                       GluAsnPheSerThrSerPheTrpIleArgIleProLysTyrPheAsn                               859095                                                                         SerIleSerLeuAsnAsnGluTyrThrIleIleAsnCysMetGluAsn                               100105110                                                                      AsnSerGlyTrpLysValSerLeuAsnTyrGlyGluIleIleTrpThr                               115120125                                                                      LeuGlnAspThrGlnGluIleLysGlnArgValValPheLysTyrSer                               130135140                                                                      GlnMetIleAsnIleSerAspTyrIleAsnArgTrpIlePheValThr                               145150155160                                                                   IleThrAsnAsnArgLeuAsnAsnSerLysIleTyrIleAsnGlyArg                               165170175                                                                      LeuIleAspGlnLysProIleSerAsnLeuGlyAsnIleHisAlaSer                               180185190                                                                      AsnAsnIleMetPheLysLeuAspGlyCysArgAspThrHisArgTyr                               195200205                                                                      IleTrpIleLysTyrPheAsnLeuPheAspLysGluLeuAsnGluLys                               210215220                                                                      GluIleLysAspLeuTyrAspAsnGlnSerAsnSerGlyIleLeuLys                               225230235240                                                                   AspPheTrpGlyAspTyrLeuGlnTyrAspLysProTyrTyrMetLeu                               245250255                                                                      AsnLeuTyrAspProAsnLysTyrValAspValAsnAsnValGlyIle                               260265270                                                                      ArgGlyTyrMetTyrLeuLysGlyProArgGlySerValMetThrThr                               275280285                                                                      AsnIleTyrLeuAsnSerSerLeuTyrArgGlyThrLysPheIleIle                               290295300                                                                      LysLysTyrAlaSerGlyAsnLysAspAsnIleValArgAsnAsnAsp                               305310315320                                                                   ArgValTyrIleAsnValValValLysAsnLysGluTyrArgLeuAla                               325330335                                                                      ThrAsnAlaSerGlnAlaGlyValGluLysIleLeuSerAlaLeuGlu                               340345350                                                                      IleProAspValGlyAsnLeuSerGlnValValValMetLysSerLys                               355360365                                                                      AsnAspGlnGlyIleThrAsnLysCysLysMetAsnLeuGlnAspAsn                               370375380                                                                      AsnGlyAsnAspIleGlyPheIleGlyPheHisGlnPheAsnAsnIle                               385390395400                                                                   AlaLysLeuValAlaSerAsnTrpTyrAsnArgGlnIleGluArgSer                               405410415                                                                      SerArgThrLeuGlyCysSerTrpGluPheIleProValAspAspGly                               420425430                                                                      TrpGlyGluArgProLeu                                                             435                                                                            (2) INFORMATION FOR SEQ ID NO:24:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 23 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: unknown                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                       MetGlyHisHisHisHisHisHisHisHisHisHisSerSerGlyHis                               151015                                                                         IleGluGlyArgHisMetAla                                                          20                                                                             (2) INFORMATION FOR SEQ ID NO:25:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1402 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..1386                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                       ATGGGCCATCATCATCATCATCATCATCATCATCACAGCAGCGGCCAT48                             MetGlyHisHisHisHisHisHisHisHisHisHisSerSerGlyHis                               151015                                                                         ATCGAAGGTCGTCATATGGCTAGCATGGCTCGTCTGCTGTCTACCTTC96                             IleGluGlyArgHisMetAlaSerMetAlaArgLeuLeuSerThrPhe                               202530                                                                         ACTGAATACATCAAGAACATCATCAATACCTCCATCCTGAACCTGCGC144                            ThrGluTyrIleLysAsnIleIleAsnThrSerIleLeuAsnLeuArg                               354045                                                                         TACGAATCCAATCACCTGATCGACCTGTCTCGCTACGCTTCCAAAATC192                            TyrGluSerAsnHisLeuIleAspLeuSerArgTyrAlaSerLysIle                               505560                                                                         AACATCGGTTCTAAAGTTAACTTCGATCCGATCGACAAGAATCAGATC240                            AsnIleGlySerLysValAsnPheAspProIleAspLysAsnGlnIle                               65707580                                                                       CAGCTGTTCAATCTGGAATCTTCCAAAATCGAAGTTATCCTGAAGAAT288                            GlnLeuPheAsnLeuGluSerSerLysIleGluValIleLeuLysAsn                               859095                                                                         GCTATCGTATACAACTCTATGTACGAAAACTTCTCCACCTCCTTCTGG336                            AlaIleValTyrAsnSerMetTyrGluAsnPheSerThrSerPheTrp                               100105110                                                                      ATCCGTATCCCGAAATACTTCAACTCCATCTCTCTGAACAATGAATAC384                            IleArgIleProLysTyrPheAsnSerIleSerLeuAsnAsnGluTyr                               115120125                                                                      ACCATCATCAACTGCATGGAAAACAATTCTGGTTGGAAAGTATCTCTG432                            ThrIleIleAsnCysMetGluAsnAsnSerGlyTrpLysValSerLeu                               130135140                                                                      AACTACGGTGAAATCATCTGGACTCTGCAGGACACTCAGGAAATCAAA480                            AsnTyrGlyGluIleIleTrpThrLeuGlnAspThrGlnGluIleLys                               145150155160                                                                   CAGCGTGTTGTATTCAAATACTCTCAGATGATCAACATCTCTGACTAC528                            GlnArgValValPheLysTyrSerGlnMetIleAsnIleSerAspTyr                               165170175                                                                      ATCAATCGCTGGATCTTCGTTACCATCACCAACAATCGTCTGAATAAC576                            IleAsnArgTrpIlePheValThrIleThrAsnAsnArgLeuAsnAsn                               180185190                                                                      TCCAAAATCTACATCAACGGCCGTCTGATCGACCAGAAACCGATCTCC624                            SerLysIleTyrIleAsnGlyArgLeuIleAspGlnLysProIleSer                               195200205                                                                      AATCTGGGTAACATCCACGCTTCTAATAACATCATGTTCAAACTGGAC672                            AsnLeuGlyAsnIleHisAlaSerAsnAsnIleMetPheLysLeuAsp                               210215220                                                                      GGTTGTCGTGACACTCACCGCTACATCTGGATCAAATACTTCAATCTG720                            GlyCysArgAspThrHisArgTyrIleTrpIleLysTyrPheAsnLeu                               225230235240                                                                   TTCGACAAAGAACTGAACGAAAAAGAAATCAAAGACCTGTACGACAAC768                            PheAspLysGluLeuAsnGluLysGluIleLysAspLeuTyrAspAsn                               245250255                                                                      CAGTCCAATTCTGGTATCCTGAAAGACTTCTGGGGTGACTACCTGCAG816                            GlnSerAsnSerGlyIleLeuLysAspPheTrpGlyAspTyrLeuGln                               260265270                                                                      TACGACAAACCGTACTACATGCTGAATCTGTACGATCCGAACAAATAC864                            TyrAspLysProTyrTyrMetLeuAsnLeuTyrAspProAsnLysTyr                               275280285                                                                      GTTGACGTCAACAATGTAGGTATCCGCGGTTACATGTACCTGAAAGGT912                            ValAspValAsnAsnValGlyIleArgGlyTyrMetTyrLeuLysGly                               290295300                                                                      CCGCGTGGTTCTGTTATGACTACCAACATCTACCTGAACTCTTCCCTG960                            ProArgGlySerValMetThrThrAsnIleTyrLeuAsnSerSerLeu                               305310315320                                                                   TACCGTGGTACCAAATTCATCATCAAGAAATACGCGTCTGGTAACAAG1008                           TyrArgGlyThrLysPheIleIleLysLysTyrAlaSerGlyAsnLys                               325330335                                                                      GACAATATCGTTCGCAACAATGATCGTGTATACATCAATGTTGTAGTT1056                           AspAsnIleValArgAsnAsnAspArgValTyrIleAsnValValVal                               340345350                                                                      AAGAACAAAGAATACCGTCTGGCTACCAATGCTTCTCAGGCTGGTGTA1104                           LysAsnLysGluTyrArgLeuAlaThrAsnAlaSerGlnAlaGlyVal                               355360365                                                                      GAAAAGATCTTGTCTGCTCTGGAAATCCCGGACGTTGGTAATCTGTCT1152                           GluLysIleLeuSerAlaLeuGluIleProAspValGlyAsnLeuSer                               370375380                                                                      CAGGTAGTTGTAATGAAATCCAAGAACGACCAGGGTATCACTAACAAA1200                           GlnValValValMetLysSerLysAsnAspGlnGlyIleThrAsnLys                               385390395400                                                                   TGCAAAATGAATCTGCAGGACAACAATGGTAACGATATCGGTTTCATC1248                           CysLysMetAsnLeuGlnAspAsnAsnGlyAsnAspIleGlyPheIle                               405410415                                                                      GGTTTCCACCAGTTCAACAATATCGCTAAACTGGTTGCTTCCAACTGG1296                           GlyPheHisGlnPheAsnAsnIleAlaLysLeuValAlaSerAsnTrp                               420425430                                                                      TACAATCGTCAGATCGAACGTTCCTCTCGCACTCTGGGTTGCTCTTGG1344                           TyrAsnArgGlnIleGluArgSerSerArgThrLeuGlyCysSerTrp                               435440445                                                                      GAGTTCATCCCGGTTGATGACGGTTGGGGTGAACGTCCGCTG1386                                 GluPheIleProValAspAspGlyTrpGlyGluArgProLeu                                     450455460                                                                      TAACCCGGGAAAGCTT1402                                                           (2) INFORMATION FOR SEQ ID NO:26:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 462 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                       MetGlyHisHisHisHisHisHisHisHisHisHisSerSerGlyHis                               151015                                                                         IleGluGlyArgHisMetAlaSerMetAlaArgLeuLeuSerThrPhe                               202530                                                                         ThrGluTyrIleLysAsnIleIleAsnThrSerIleLeuAsnLeuArg                               354045                                                                         TyrGluSerAsnHisLeuIleAspLeuSerArgTyrAlaSerLysIle                               505560                                                                         AsnIleGlySerLysValAsnPheAspProIleAspLysAsnGlnIle                               65707580                                                                       GlnLeuPheAsnLeuGluSerSerLysIleGluValIleLeuLysAsn                               859095                                                                         AlaIleValTyrAsnSerMetTyrGluAsnPheSerThrSerPheTrp                               100105110                                                                      IleArgIleProLysTyrPheAsnSerIleSerLeuAsnAsnGluTyr                               115120125                                                                      ThrIleIleAsnCysMetGluAsnAsnSerGlyTrpLysValSerLeu                               130135140                                                                      AsnTyrGlyGluIleIleTrpThrLeuGlnAspThrGlnGluIleLys                               145150155160                                                                   GlnArgValValPheLysTyrSerGlnMetIleAsnIleSerAspTyr                               165170175                                                                      IleAsnArgTrpIlePheValThrIleThrAsnAsnArgLeuAsnAsn                               180185190                                                                      SerLysIleTyrIleAsnGlyArgLeuIleAspGlnLysProIleSer                               195200205                                                                      AsnLeuGlyAsnIleHisAlaSerAsnAsnIleMetPheLysLeuAsp                               210215220                                                                      GlyCysArgAspThrHisArgTyrIleTrpIleLysTyrPheAsnLeu                               225230235240                                                                   PheAspLysGluLeuAsnGluLysGluIleLysAspLeuTyrAspAsn                               245250255                                                                      GlnSerAsnSerGlyIleLeuLysAspPheTrpGlyAspTyrLeuGln                               260265270                                                                      TyrAspLysProTyrTyrMetLeuAsnLeuTyrAspProAsnLysTyr                               275280285                                                                      ValAspValAsnAsnValGlyIleArgGlyTyrMetTyrLeuLysGly                               290295300                                                                      ProArgGlySerValMetThrThrAsnIleTyrLeuAsnSerSerLeu                               305310315320                                                                   TyrArgGlyThrLysPheIleIleLysLysTyrAlaSerGlyAsnLys                               325330335                                                                      AspAsnIleValArgAsnAsnAspArgValTyrIleAsnValValVal                               340345350                                                                      LysAsnLysGluTyrArgLeuAlaThrAsnAlaSerGlnAlaGlyVal                               355360365                                                                      GluLysIleLeuSerAlaLeuGluIleProAspValGlyAsnLeuSer                               370375380                                                                      GlnValValValMetLysSerLysAsnAspGlnGlyIleThrAsnLys                               385390395400                                                                   CysLysMetAsnLeuGlnAspAsnAsnGlyAsnAspIleGlyPheIle                               405410415                                                                      GlyPheHisGlnPheAsnAsnIleAlaLysLeuValAlaSerAsnTrp                               420425430                                                                      TyrAsnArgGlnIleGluArgSerSerArgThrLeuGlyCysSerTrp                               435440445                                                                      GluPheIleProValAspAspGlyTrpGlyGluArgProLeu                                     450455460                                                                      (2) INFORMATION FOR SEQ ID NO:27:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 3891 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..3888                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                       ATGCAATTTGTTAATAAACAATTTAATTATAAAGATCCTGTAAATGGT48                             MetGlnPheValAsnLysGlnPheAsnTyrLysAspProValAsnGly                               151015                                                                         GTTGATATTGCTTATATAAAAATTCCAAATGTAGGACAAATGCAACCA96                             ValAspIleAlaTyrIleLysIleProAsnValGlyGlnMetGlnPro                               202530                                                                         GTAAAAGCTTTTAAAATTCATAATAAAATATGGGTTATTCCAGAAAGA144                            ValLysAlaPheLysIleHisAsnLysIleTrpValIleProGluArg                               354045                                                                         GATACATTTACAAATCCTGAAGAAGGAGATTTAAATCCACCACCAGAA192                            AspThrPheThrAsnProGluGluGlyAspLeuAsnProProProGlu                               505560                                                                         GCAAAACAAGTTCCAGTTTCATATTATGATTCAACATATTTAAGTACA240                            AlaLysGlnValProValSerTyrTyrAspSerThrTyrLeuSerThr                               65707580                                                                       GATAATGAAAAAGATAATTATTTAAAGGGAGTTACAAAATTATTTGAG288                            AspAsnGluLysAspAsnTyrLeuLysGlyValThrLysLeuPheGlu                               859095                                                                         AGAATTTATTCAACTGATCTTGGAAGAATGTTGTTAACATCAATAGTA336                            ArgIleTyrSerThrAspLeuGlyArgMetLeuLeuThrSerIleVal                               100105110                                                                      AGGGGAATACCATTTTGGGGTGGAAGTACAATAGATACAGAATTAAAA384                            ArgGlyIleProPheTrpGlyGlySerThrIleAspThrGluLeuLys                               115120125                                                                      GTTATTGATACTAATTGTATTAATGTGATACAACCAGATGGTAGTTAT432                            ValIleAspThrAsnCysIleAsnValIleGlnProAspGlySerTyr                               130135140                                                                      AGATCAGAAGAACTTAATCTAGTAATAATAGGACCCTCAGCTGATATT480                            ArgSerGluGluLeuAsnLeuValIleIleGlyProSerAlaAspIle                               145150155160                                                                   ATACAGTTTGAATGTAAAAGCTTTGGACATGAAGTTTTGAATCTTACG528                            IleGlnPheGluCysLysSerPheGlyHisGluValLeuAsnLeuThr                               165170175                                                                      CGAAATGGTTATGGCTCTACTCAATACATTAGATTTAGCCCAGATTTT576                            ArgAsnGlyTyrGlySerThrGlnTyrIleArgPheSerProAspPhe                               180185190                                                                      ACATTTGGTTTTGAGGAGTCACTTGAAGTTGATACAAATCCTCTTTTA624                            ThrPheGlyPheGluGluSerLeuGluValAspThrAsnProLeuLeu                               195200205                                                                      GGTGCAGGCAAATTTGCTACAGATCCAGCAGTAACATTAGCACATGAA672                            GlyAlaGlyLysPheAlaThrAspProAlaValThrLeuAlaHisGlu                               210215220                                                                      CTTATACATGCTGGACATAGATTATATGGAATAGCAATTAATCCAAAT720                            LeuIleHisAlaGlyHisArgLeuTyrGlyIleAlaIleAsnProAsn                               225230235240                                                                   AGGGTTTTTAAAGTAAATACTAATGCCTATTATGAAATGAGTGGGTTA768                            ArgValPheLysValAsnThrAsnAlaTyrTyrGluMetSerGlyLeu                               245250255                                                                      GAAGTAAGCTTTGAGGAACTTAGAACATTTGGGGGACATGATGCAAAG816                            GluValSerPheGluGluLeuArgThrPheGlyGlyHisAspAlaLys                               260265270                                                                      TTTATAGATAGTTTACAGGAAAACGAATTTCGTCTATATTATTATAAT864                            PheIleAspSerLeuGlnGluAsnGluPheArgLeuTyrTyrTyrAsn                               275280285                                                                      AAGTTTAAAGATATAGCAAGTACACTTAATAAAGCTAAATCAATAGTA912                            LysPheLysAspIleAlaSerThrLeuAsnLysAlaLysSerIleVal                               290295300                                                                      GGTACTACTGCTTCATTACAGTATATGAAAAATGTTTTTAAAGAGAAA960                            GlyThrThrAlaSerLeuGlnTyrMetLysAsnValPheLysGluLys                               305310315320                                                                   TATCTCCTATCTGAAGATACATCTGGAAAATTTTCGGTAGATAAATTA1008                           TyrLeuLeuSerGluAspThrSerGlyLysPheSerValAspLysLeu                               325330335                                                                      AAATTTGATAAGTTATACAAAATGTTAACAGAGATTTACACAGAGGAT1056                           LysPheAspLysLeuTyrLysMetLeuThrGluIleTyrThrGluAsp                               340345350                                                                      AATTTTGTTAAGTTTTTTAAAGTACTTAACAGAAAAACATATTTGAAT1104                           AsnPheValLysPhePheLysValLeuAsnArgLysThrTyrLeuAsn                               355360365                                                                      TTTGATAAAGCCGTATTTAAGATAAATATAGTACCTAAGGTAAATTAC1152                           PheAspLysAlaValPheLysIleAsnIleValProLysValAsnTyr                               370375380                                                                      ACAATATATGATGGATTTAATTTAAGAAATACAAATTTAGCAGCAAAC1200                           ThrIleTyrAspGlyPheAsnLeuArgAsnThrAsnLeuAlaAlaAsn                               385390395400                                                                   TTTAATGGTCAAAATACAGAAATTAATAATATGAATTTTACTAAACTA1248                           PheAsnGlyGlnAsnThrGluIleAsnAsnMetAsnPheThrLysLeu                               405410415                                                                      AAAAATTTTACTGGATTGTTTGAATTTTATAAGTTGCTATGTGTAAGA1296                           LysAsnPheThrGlyLeuPheGluPheTyrLysLeuLeuCysValArg                               420425430                                                                      GGGATAATAACTTCTAAAACTAAATCATTAGATAAAGGATACAATAAG1344                           GlyIleIleThrSerLysThrLysSerLeuAspLysGlyTyrAsnLys                               435440445                                                                      GCATTAAATGATTTATGTATCAAAGTTAATAATTGGGACTTGTTTTTT1392                           AlaLeuAsnAspLeuCysIleLysValAsnAsnTrpAspLeuPhePhe                               450455460                                                                      AGTCCTTCAGAAGATAATTTTACTAATGATCTAAATAAAGGAGAAGAA1440                           SerProSerGluAspAsnPheThrAsnAspLeuAsnLysGlyGluGlu                               465470475480                                                                   ATTACATCTGATACTAATATAGAAGCAGCAGAAGAAAATATTAGTTTA1488                           IleThrSerAspThrAsnIleGluAlaAlaGluGluAsnIleSerLeu                               485490495                                                                      GATTTAATACAACAATATTATTTAACCTTTAATTTTGATAATGAACCT1536                           AspLeuIleGlnGlnTyrTyrLeuThrPheAsnPheAspAsnGluPro                               500505510                                                                      GAAAATATTTCAATAGAAAATCTTTCAAGTGACATTATAGGCCAATTA1584                           GluAsnIleSerIleGluAsnLeuSerSerAspIleIleGlyGlnLeu                               515520525                                                                      GAACTTATGCCTAATATAGAAAGATTTCCTAATGGAAAAAAGTATGAG1632                           GluLeuMetProAsnIleGluArgPheProAsnGlyLysLysTyrGlu                               530535540                                                                      TTAGATAAATATACTATGTTCCATTATCTTCGTGCTCAAGAATTTGAA1680                           LeuAspLysTyrThrMetPheHisTyrLeuArgAlaGlnGluPheGlu                               545550555560                                                                   CATGGTAAATCTAGGATTGCTTTAACAAATTCTGTTAACGAAGCATTA1728                           HisGlyLysSerArgIleAlaLeuThrAsnSerValAsnGluAlaLeu                               565570575                                                                      TTAAATCCTAGTCGTGTTTATACATTTTTTTCTTCAGACTATGTAAAG1776                           LeuAsnProSerArgValTyrThrPhePheSerSerAspTyrValLys                               580585590                                                                      AAAGTTAATAAAGCTACGGAGGCAGCTATGTTTTTAGGCTGGGTAGAA1824                           LysValAsnLysAlaThrGluAlaAlaMetPheLeuGlyTrpValGlu                               595600605                                                                      CAATTAGTATATGATTTTACCGATGAAACTAGCGAAGTAAGTACTACG1872                           GlnLeuValTyrAspPheThrAspGluThrSerGluValSerThrThr                               610615620                                                                      GATAAAATTGCGGATATAACTATAATTATTCCATATATAGGACCTGCT1920                           AspLysIleAlaAspIleThrIleIleIleProTyrIleGlyProAla                               625630635640                                                                   TTAAATATAGGTAATATGTTATATAAAGATGATTTTGTAGGTGCTTTA1968                           LeuAsnIleGlyAsnMetLeuTyrLysAspAspPheValGlyAlaLeu                               645650655                                                                      ATATTTTCAGGAGCTGTTATTCTGTTAGAATTTATACCAGAGATTGCA2016                           IlePheSerGlyAlaValIleLeuLeuGluPheIleProGluIleAla                               660665670                                                                      ATACCTGTATTAGGTACTTTTGCACTTGTATCATATATTGCGAATAAG2064                           IleProValLeuGlyThrPheAlaLeuValSerTyrIleAlaAsnLys                               675680685                                                                      GTTCTAACCGTTCAAACAATAGATAATGCTTTAAGTAAAAGAAATGAA2112                           ValLeuThrValGlnThrIleAspAsnAlaLeuSerLysArgAsnGlu                               690695700                                                                      AAATGGGATGAGGTCTATAAATATATAGTAACAAATTGGTTAGCAAAG2160                           LysTrpAspGluValTyrLysTyrIleValThrAsnTrpLeuAlaLys                               705710715720                                                                   GTTAATACACAGATTGATCTAATAAGAAAAAAAATGAAAGAAGCTTTA2208                           ValAsnThrGlnIleAspLeuIleArgLysLysMetLysGluAlaLeu                               725730735                                                                      GAAAATCAAGCAGAAGCAACAAAGGCTATAATAAACTATCAGTATAAT2256                           GluAsnGlnAlaGluAlaThrLysAlaIleIleAsnTyrGlnTyrAsn                               740745750                                                                      CAATATACTGAGGAAGAGAAAAATAATATTAATTTTAATATTGATGAT2304                           GlnTyrThrGluGluGluLysAsnAsnIleAsnPheAsnIleAspAsp                               755760765                                                                      TTAAGTTCGAAACTTAATGAGTCTATAAATAAAGCTATGATTAATATA2352                           LeuSerSerLysLeuAsnGluSerIleAsnLysAlaMetIleAsnIle                               770775780                                                                      AATAAATTTTTGAATCAATGCTCTGTTTCATATTTAATGAATTCTATG2400                           AsnLysPheLeuAsnGlnCysSerValSerTyrLeuMetAsnSerMet                               785790795800                                                                   ATCCCTTATGGTGTTAAACGGTTAGAAGATTTTGATGCTAGTCTTAAA2448                           IleProTyrGlyValLysArgLeuGluAspPheAspAlaSerLeuLys                               805810815                                                                      GATGCATTATTAAAGTATATATATGATAATAGAGGAACTTTAATTGGT2496                           AspAlaLeuLeuLysTyrIleTyrAspAsnArgGlyThrLeuIleGly                               820825830                                                                      CAAGTAGATAGATTAAAAGATAAAGTTAATAATACACTTAGTACAGAT2544                           GlnValAspArgLeuLysAspLysValAsnAsnThrLeuSerThrAsp                               835840845                                                                      ATACCTTTTCAGCTTTCCAAATACGTAGATAATCAAAGATTATTATCT2592                           IleProPheGlnLeuSerLysTyrValAspAsnGlnArgLeuLeuSer                               850855860                                                                      ACATTTACTGAATATATTAAGAATATTATTAATACTTCTATATTGAAT2640                           ThrPheThrGluTyrIleLysAsnIleIleAsnThrSerIleLeuAsn                               865870875880                                                                   TTAAGATATGAAAGTAATCATTTAATAGACTTATCTAGGTATGCATCA2688                           LeuArgTyrGluSerAsnHisLeuIleAspLeuSerArgTyrAlaSer                               885890895                                                                      AAAATAAATATTGGTAGTAAAGTAAATTTTGATCCAATAGATAAAAAT2736                           LysIleAsnIleGlySerLysValAsnPheAspProIleAspLysAsn                               900905910                                                                      CAAATTCAATTATTTAATTTAGAAAGTAGTAAAATTGAGGTAATTTTA2784                           GlnIleGlnLeuPheAsnLeuGluSerSerLysIleGluValIleLeu                               915920925                                                                      AAAAATGCTATTGTATATAATAGTATGTATGAAAATTTTAGTACTAGC2832                           LysAsnAlaIleValTyrAsnSerMetTyrGluAsnPheSerThrSer                               930935940                                                                      TTTTGGATAAGAATTCCTAAGTATTTTAACAGTATAAGTCTAAATAAT2880                           PheTrpIleArgIleProLysTyrPheAsnSerIleSerLeuAsnAsn                               945950955960                                                                   GAATATACAATAATAAATTGTATGGAAAATAATTCAGGATGGAAAGTA2928                           GluTyrThrIleIleAsnCysMetGluAsnAsnSerGlyTrpLysVal                               965970975                                                                      TCACTTAATTATGGTGAAATAATCTGGACTTTACAGGATACTCAGGAA2976                           SerLeuAsnTyrGlyGluIleIleTrpThrLeuGlnAspThrGlnGlu                               980985990                                                                      ATAAAACAAAGAGTAGTTTTTAAATACAGTCAAATGATTAATATATCA3024                           IleLysGlnArgValValPheLysTyrSerGlnMetIleAsnIleSer                               99510001005                                                                    GATTATATAAACAGATGGATTTTTGTAACTATCACTAATAATAGATTA3072                           AspTyrIleAsnArgTrpIlePheValThrIleThrAsnAsnArgLeu                               101010151020                                                                   AATAACTCTAAAATTTATATAAATGGAAGATTAATAGATCAAAAACCA3120                           AsnAsnSerLysIleTyrIleAsnGlyArgLeuIleAspGlnLysPro                               1025103010351040                                                               ATTTCAAATTTAGGTAATATTCATGCTAGTAATAATATAATGTTTAAA3168                           IleSerAsnLeuGlyAsnIleHisAlaSerAsnAsnIleMetPheLys                               104510501055                                                                   TTAGATGGTTGTAGAGATACACATAGATATATTTGGATAAAATATTTT3216                           LeuAspGlyCysArgAspThrHisArgTyrIleTrpIleLysTyrPhe                               106010651070                                                                   AATCTTTTTGATAAGGAATTAAATGAAAAAGAAATCAAAGATTTATAT3264                           AsnLeuPheAspLysGluLeuAsnGluLysGluIleLysAspLeuTyr                               107510801085                                                                   GATAATCAATCAAATTCAGGTATTTTAAAAGACTTTTGGGGTGATTAT3312                           AspAsnGlnSerAsnSerGlyIleLeuLysAspPheTrpGlyAspTyr                               109010951100                                                                   TTACAATATGATAAACCATACTATATGTTAAATTTATATGATCCAAAT3360                           LeuGlnTyrAspLysProTyrTyrMetLeuAsnLeuTyrAspProAsn                               1105111011151120                                                               AAATATGTCGATGTAAATAATGTAGGTATTAGAGGTTATATGTATCTT3408                           LysTyrValAspValAsnAsnValGlyIleArgGlyTyrMetTyrLeu                               112511301135                                                                   AAAGGGCCTAGAGGTAGCGTAATGACTACAAACATTTATTTAAATTCA3456                           LysGlyProArgGlySerValMetThrThrAsnIleTyrLeuAsnSer                               114011451150                                                                   AGTTTGTATAGGGGGACAAAATTTATTATAAAAAAATATGCTTCTGGA3504                           SerLeuTyrArgGlyThrLysPheIleIleLysLysTyrAlaSerGly                               115511601165                                                                   AATAAAGATAATATTGTTAGAAATAATGATCGTGTATATATTAATGTA3552                           AsnLysAspAsnIleValArgAsnAsnAspArgValTyrIleAsnVal                               117011751180                                                                   GTAGTTAAAAATAAAGAATATAGGTTAGCTACTAATGCATCACAGGCA3600                           ValValLysAsnLysGluTyrArgLeuAlaThrAsnAlaSerGlnAla                               1185119011951200                                                               GGCGTAGAAAAAATACTAAGTGCATTAGAAATACCTGATGTAGGAAAT3648                           GlyValGluLysIleLeuSerAlaLeuGluIleProAspValGlyAsn                               120512101215                                                                   CTAAGTCAAGTAGTAGTAATGAAGTCAAAAAATGATCAAGGAATAACA3696                           LeuSerGlnValValValMetLysSerLysAsnAspGlnGlyIleThr                               122012251230                                                                   AATAAATGCAAAATGAATTTACAAGATAATAATGGGAATGATATAGGC3744                           AsnLysCysLysMetAsnLeuGlnAspAsnAsnGlyAsnAspIleGly                               123512401245                                                                   TTTATAGGATTTCATCAGTTTAATAATATAGCTAAACTAGTAGCAAGT3792                           PheIleGlyPheHisGlnPheAsnAsnIleAlaLysLeuValAlaSer                               125012551260                                                                   AATTGGTATAATAGACAAATAGAAAGATCTAGTAGGACTTTGGGTTGC3840                           AsnTrpTyrAsnArgGlnIleGluArgSerSerArgThrLeuGlyCys                               1265127012751280                                                               TCATGGGAATTTATTCCTGTAGATGATGGATGGGGAGAAAGGCCACTG3888                           SerTrpGluPheIleProValAspAspGlyTrpGlyGluArgProLeu                               128512901295                                                                   TAA3891                                                                        (2) INFORMATION FOR SEQ ID NO:28:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1296 amino acids                                                   (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                       MetGlnPheValAsnLysGlnPheAsnTyrLysAspProValAsnGly                               151015                                                                         ValAspIleAlaTyrIleLysIleProAsnValGlyGlnMetGlnPro                               202530                                                                         ValLysAlaPheLysIleHisAsnLysIleTrpValIleProGluArg                               354045                                                                         AspThrPheThrAsnProGluGluGlyAspLeuAsnProProProGlu                               505560                                                                         AlaLysGlnValProValSerTyrTyrAspSerThrTyrLeuSerThr                               65707580                                                                       AspAsnGluLysAspAsnTyrLeuLysGlyValThrLysLeuPheGlu                               859095                                                                         ArgIleTyrSerThrAspLeuGlyArgMetLeuLeuThrSerIleVal                               100105110                                                                      ArgGlyIleProPheTrpGlyGlySerThrIleAspThrGluLeuLys                               115120125                                                                      ValIleAspThrAsnCysIleAsnValIleGlnProAspGlySerTyr                               130135140                                                                      ArgSerGluGluLeuAsnLeuValIleIleGlyProSerAlaAspIle                               145150155160                                                                   IleGlnPheGluCysLysSerPheGlyHisGluValLeuAsnLeuThr                               165170175                                                                      ArgAsnGlyTyrGlySerThrGlnTyrIleArgPheSerProAspPhe                               180185190                                                                      ThrPheGlyPheGluGluSerLeuGluValAspThrAsnProLeuLeu                               195200205                                                                      GlyAlaGlyLysPheAlaThrAspProAlaValThrLeuAlaHisGlu                               210215220                                                                      LeuIleHisAlaGlyHisArgLeuTyrGlyIleAlaIleAsnProAsn                               225230235240                                                                   ArgValPheLysValAsnThrAsnAlaTyrTyrGluMetSerGlyLeu                               245250255                                                                      GluValSerPheGluGluLeuArgThrPheGlyGlyHisAspAlaLys                               260265270                                                                      PheIleAspSerLeuGlnGluAsnGluPheArgLeuTyrTyrTyrAsn                               275280285                                                                      LysPheLysAspIleAlaSerThrLeuAsnLysAlaLysSerIleVal                               290295300                                                                      GlyThrThrAlaSerLeuGlnTyrMetLysAsnValPheLysGluLys                               305310315320                                                                   TyrLeuLeuSerGluAspThrSerGlyLysPheSerValAspLysLeu                               325330335                                                                      LysPheAspLysLeuTyrLysMetLeuThrGluIleTyrThrGluAsp                               340345350                                                                      AsnPheValLysPhePheLysValLeuAsnArgLysThrTyrLeuAsn                               355360365                                                                      PheAspLysAlaValPheLysIleAsnIleValProLysValAsnTyr                               370375380                                                                      ThrIleTyrAspGlyPheAsnLeuArgAsnThrAsnLeuAlaAlaAsn                               385390395400                                                                   PheAsnGlyGlnAsnThrGluIleAsnAsnMetAsnPheThrLysLeu                               405410415                                                                      LysAsnPheThrGlyLeuPheGluPheTyrLysLeuLeuCysValArg                               420425430                                                                      GlyIleIleThrSerLysThrLysSerLeuAspLysGlyTyrAsnLys                               435440445                                                                      AlaLeuAsnAspLeuCysIleLysValAsnAsnTrpAspLeuPhePhe                               450455460                                                                      SerProSerGluAspAsnPheThrAsnAspLeuAsnLysGlyGluGlu                               465470475480                                                                   IleThrSerAspThrAsnIleGluAlaAlaGluGluAsnIleSerLeu                               485490495                                                                      AspLeuIleGlnGlnTyrTyrLeuThrPheAsnPheAspAsnGluPro                               500505510                                                                      GluAsnIleSerIleGluAsnLeuSerSerAspIleIleGlyGlnLeu                               515520525                                                                      GluLeuMetProAsnIleGluArgPheProAsnGlyLysLysTyrGlu                               530535540                                                                      LeuAspLysTyrThrMetPheHisTyrLeuArgAlaGlnGluPheGlu                               545550555560                                                                   HisGlyLysSerArgIleAlaLeuThrAsnSerValAsnGluAlaLeu                               565570575                                                                      LeuAsnProSerArgValTyrThrPhePheSerSerAspTyrValLys                               580585590                                                                      LysValAsnLysAlaThrGluAlaAlaMetPheLeuGlyTrpValGlu                               595600605                                                                      GlnLeuValTyrAspPheThrAspGluThrSerGluValSerThrThr                               610615620                                                                      AspLysIleAlaAspIleThrIleIleIleProTyrIleGlyProAla                               625630635640                                                                   LeuAsnIleGlyAsnMetLeuTyrLysAspAspPheValGlyAlaLeu                               645650655                                                                      IlePheSerGlyAlaValIleLeuLeuGluPheIleProGluIleAla                               660665670                                                                      IleProValLeuGlyThrPheAlaLeuValSerTyrIleAlaAsnLys                               675680685                                                                      ValLeuThrValGlnThrIleAspAsnAlaLeuSerLysArgAsnGlu                               690695700                                                                      LysTrpAspGluValTyrLysTyrIleValThrAsnTrpLeuAlaLys                               705710715720                                                                   ValAsnThrGlnIleAspLeuIleArgLysLysMetLysGluAlaLeu                               725730735                                                                      GluAsnGlnAlaGluAlaThrLysAlaIleIleAsnTyrGlnTyrAsn                               740745750                                                                      GlnTyrThrGluGluGluLysAsnAsnIleAsnPheAsnIleAspAsp                               755760765                                                                      LeuSerSerLysLeuAsnGluSerIleAsnLysAlaMetIleAsnIle                               770775780                                                                      AsnLysPheLeuAsnGlnCysSerValSerTyrLeuMetAsnSerMet                               785790795800                                                                   IleProTyrGlyValLysArgLeuGluAspPheAspAlaSerLeuLys                               805810815                                                                      AspAlaLeuLeuLysTyrIleTyrAspAsnArgGlyThrLeuIleGly                               820825830                                                                      GlnValAspArgLeuLysAspLysValAsnAsnThrLeuSerThrAsp                               835840845                                                                      IleProPheGlnLeuSerLysTyrValAspAsnGlnArgLeuLeuSer                               850855860                                                                      ThrPheThrGluTyrIleLysAsnIleIleAsnThrSerIleLeuAsn                               865870875880                                                                   LeuArgTyrGluSerAsnHisLeuIleAspLeuSerArgTyrAlaSer                               885890895                                                                      LysIleAsnIleGlySerLysValAsnPheAspProIleAspLysAsn                               900905910                                                                      GlnIleGlnLeuPheAsnLeuGluSerSerLysIleGluValIleLeu                               915920925                                                                      LysAsnAlaIleValTyrAsnSerMetTyrGluAsnPheSerThrSer                               930935940                                                                      PheTrpIleArgIleProLysTyrPheAsnSerIleSerLeuAsnAsn                               945950955960                                                                   GluTyrThrIleIleAsnCysMetGluAsnAsnSerGlyTrpLysVal                               965970975                                                                      SerLeuAsnTyrGlyGluIleIleTrpThrLeuGlnAspThrGlnGlu                               980985990                                                                      IleLysGlnArgValValPheLysTyrSerGlnMetIleAsnIleSer                               99510001005                                                                    AspTyrIleAsnArgTrpIlePheValThrIleThrAsnAsnArgLeu                               101010151020                                                                   AsnAsnSerLysIleTyrIleAsnGlyArgLeuIleAspGlnLysPro                               1025103010351040                                                               IleSerAsnLeuGlyAsnIleHisAlaSerAsnAsnIleMetPheLys                               104510501055                                                                   LeuAspGlyCysArgAspThrHisArgTyrIleTrpIleLysTyrPhe                               106010651070                                                                   AsnLeuPheAspLysGluLeuAsnGluLysGluIleLysAspLeuTyr                               107510801085                                                                   AspAsnGlnSerAsnSerGlyIleLeuLysAspPheTrpGlyAspTyr                               109010951100                                                                   LeuGlnTyrAspLysProTyrTyrMetLeuAsnLeuTyrAspProAsn                               1105111011151120                                                               LysTyrValAspValAsnAsnValGlyIleArgGlyTyrMetTyrLeu                               112511301135                                                                   LysGlyProArgGlySerValMetThrThrAsnIleTyrLeuAsnSer                               114011451150                                                                   SerLeuTyrArgGlyThrLysPheIleIleLysLysTyrAlaSerGly                               115511601165                                                                   AsnLysAspAsnIleValArgAsnAsnAspArgValTyrIleAsnVal                               117011751180                                                                   ValValLysAsnLysGluTyrArgLeuAlaThrAsnAlaSerGlnAla                               1185119011951200                                                               GlyValGluLysIleLeuSerAlaLeuGluIleProAspValGlyAsn                               120512101215                                                                   LeuSerGlnValValValMetLysSerLysAsnAspGlnGlyIleThr                               122012251230                                                                   AsnLysCysLysMetAsnLeuGlnAspAsnAsnGlyAsnAspIleGly                               123512401245                                                                   PheIleGlyPheHisGlnPheAsnAsnIleAlaLysLeuValAlaSer                               125012551260                                                                   AsnTrpTyrAsnArgGlnIleGluArgSerSerArgThrLeuGlyCys                               1265127012751280                                                               SerTrpGluPheIleProValAspAspGlyTrpGlyGluArgProLeu                               128512901295                                                                   (2) INFORMATION FOR SEQ ID NO:29:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 812 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: unknown                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                       ThrSerTyrLysIleIleAsnGlyLysHisPheTyrPheAsnAsnAsp                               151015                                                                         GlyValMetGlnLeuGlyValPheLysGlyProAspGlyPheGluTyr                               202530                                                                         PheAlaProAlaAsnThrGlnAsnAsnAsnIleGluGlyGlnAlaIle                               354045                                                                         ValTyrGlnSerLysPheLeuThrLeuAsnGlyLysLysTyrTyrPhe                               505560                                                                         AspAsnAsnSerLysAlaValThrGlyTrpArgIleIleAsnAsnGlu                               65707580                                                                       LysTyrTyrPheAsnProAsnAsnAlaIleAlaAlaValGlyLeuGln                               859095                                                                         ValIleAspAsnAsnLysTyrTyrPheAsnProAspThrAlaIleIle                               100105110                                                                      SerLysGlyTrpGlnThrValAsnGlySerArgTyrTyrPheAspThr                               115120125                                                                      AspThrAlaIleAlaPheAsnGlyTyrLysThrIleAspGlyLysHis                               130135140                                                                      PheTyrPheAspSerAspCysValValLysIleGlyValPheSerThr                               145150155160                                                                   SerAsnGlyPheGluTyrPheAlaProAlaAsnThrTyrAsnAsnAsn                               165170175                                                                      IleGluGlyGlnAlaIleValTyrGlnSerLysPheLeuThrLeuAsn                               180185190                                                                      GlyLysLysTyrTyrPheAspAsnAsnSerLysAlaValThrGlyLeu                               195200205                                                                      GlnThrIleAspSerLysLysTyrTyrPheAsnThrAsnThrAlaGlu                               210215220                                                                      AlaAlaThrGlyTrpGlnThrIleAspGlyLysLysTyrTyrPheAsn                               225230235240                                                                   ThrAsnThrAlaGluAlaAlaThrGlyTrpGlnThrIleAspGlyLys                               245250255                                                                      LysTyrTyrPheAsnThrAsnThrAlaIleAlaSerThrGlyTyrThr                               260265270                                                                      IleIleAsnGlyLysHisPheTyrPheAsnThrAspGlyIleMetGln                               275280285                                                                      IleGlyValPheLysGlyProAsnGlyPheGluTyrPheAlaProAla                               290295300                                                                      AsnThrAspAlaAsnAsnIleGluGlyGlnAlaIleLeuTyrGlnAsn                               305310315320                                                                   GluPheLeuThrLeuAsnGlyLysLysTyrTyrPheGlySerAspSer                               325330335                                                                      LysAlaValThrGlyTrpArgIleIleAsnAsnLysLysTyrTyrPhe                               340345350                                                                      AsnProAsnAsnAlaIleAlaAlaIleHisLeuCysThrIleAsnAsn                               355360365                                                                      AspLysTyrTyrPheSerTyrAspGlyIleLeuGlnAsnGlyTyrIle                               370375380                                                                      ThrIleGluArgAsnAsnPheTyrPheAspAlaAsnAsnGluSerLys                               385390395400                                                                   MetValThrGlyValPheLysGlyProAsnGlyPheGluTyrPheAla                               405410415                                                                      ProAlaAsnThrHisAsnAsnAsnIleGluGlyGlnAlaIleValTyr                               420425430                                                                      GlnAsnLysPheLeuThrLeuAsnGlyLysLysTyrTyrPheAspAsn                               435440445                                                                      AspSerLysAlaValThrGlyTrpGlnThrIleAspGlyLysLysTyr                               450455460                                                                      TyrPheAsnLeuAsnThrAlaGluAlaAlaThrGlyTrpGlnThrIle                               465470475480                                                                   AspGlyLysLysTyrTyrPheAsnLeuAsnThrAlaGluAlaAlaThr                               485490495                                                                      GlyTrpGlnThrIleAspGlyLysLysTyrTyrPheAsnThrAsnThr                               500505510                                                                      PheIleAlaSerThrGlyTyrThrSerIleAsnGlyLysHisPheTyr                               515520525                                                                      PheAsnThrAspGlyIleMetGlnIleGlyValPheLysGlyProAsn                               530535540                                                                      GlyPheGluTyrPheAlaProAlaAsnThrAspAlaAsnAsnIleGlu                               545550555560                                                                   GlyGlnAlaIleLeuTyrGlnAsnLysPheLeuThrLeuAsnGlyLys                               565570575                                                                      LysTyrTyrPheGlySerAspSerLysAlaValThrGlyLeuArgThr                               580585590                                                                      IleAspGlyLysLysTyrTyrPheAsnThrAsnThrAlaValAlaVal                               595600605                                                                      ThrGlyTrpGlnThrIleAsnGlyLysLysTyrTyrPheAsnThrAsn                               610615620                                                                      ThrSerIleAlaSerThrGlyTyrThrIleIleSerGlyLysHisPhe                               625630635640                                                                   TyrPheAsnThrAspGlyIleMetGlnIleGlyValPheLysGlyPro                               645650655                                                                      AspGlyPheGluTyrPheAlaProAlaAsnThrAspAlaAsnAsnIle                               660665670                                                                      GluGlyGlnAlaIleArgTyrGlnAsnArgPheLeuTyrLeuHisAsp                               675680685                                                                      AsnIleTyrTyrPheGlyAsnAsnSerLysAlaAlaThrGlyTrpVal                               690695700                                                                      ThrIleAspGlyAsnArgTyrTyrPheGluProAsnThrAlaMetGly                               705710715720                                                                   AlaAsnGlyTyrLysThrIleAspAsnLysAsnPheTyrPheArgAsn                               725730735                                                                      GlyLeuProGlnIleGlyValPheLysGlySerAsnGlyPheGluTyr                               740745750                                                                      PheAlaProAlaAsnThrAspAlaAsnAsnIleGluGlyGlnAlaIle                               755760765                                                                      ArgTyrGlnAsnArgPheLeuHisLeuLeuGlyLysIleTyrTyrPhe                               770775780                                                                      GlyAsnAsnSerLysAlaValThrGlyTrpGlnThrIleAsnGlyLys                               785790795800                                                                   ValTyrTyrPheMetProAspThrAlaMetAlaAla                                           805810                                                                         (2) INFORMATION FOR SEQ ID NO:30:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 609 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: unknown                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                       ThrSerGluGluAsnLysValSerGlnValLysIleArgPheValAsn                               151015                                                                         ValPheLysAspLysThrLeuAlaAsnLysLeuSerPheAsnPheSer                               202530                                                                         AspLysGlnAspValProValSerGluIleIleLeuSerPheThrPro                               354045                                                                         SerTyrTyrGluAspGlyLeuIleGlyTyrAspLeuGlyLeuValSer                               505560                                                                         LeuTyrAsnGluLysPheTyrIleAsnAsnPheGlyMetMetValSer                               65707580                                                                       GlyLeuIleTyrIleAsnAspSerLeuTyrTyrPheLysProProVal                               859095                                                                         AsnAsnLeuIleThrGlyPheValThrValGlyAspAspLysTyrTyr                               100105110                                                                      PheAsnProIleAsnGlyGlyAlaAlaSerIleGlyGluThrIleIle                               115120125                                                                      AspAspLysAsnTyrTyrPheAsnGlnSerGlyValLeuGlnThrGly                               130135140                                                                      ValPheSerThrGluAspGlyPheLysTyrPheAlaProAlaAsnThr                               145150155160                                                                   LeuAspGluAsnLeuGluGlyGluAlaIleAspPheThrGlyLysLeu                               165170175                                                                      IleIleAspGluAsnIleTyrTyrPheAspAspAsnTyrArgGlyAla                               180185190                                                                      ValGluTrpLysGluLeuAspGlyGluMetHisTyrPheSerProGlu                               195200205                                                                      ThrGlyLysAlaPheLysGlyLeuAsnGlnIleGlyAspTyrLysTyr                               210215220                                                                      TyrPheAsnSerAspGlyValMetGlnLysGlyPheValSerIleAsn                               225230235240                                                                   AspAsnLysHisTyrPheAspAspSerGlyValMetLysValGlyTyr                               245250255                                                                      ThrGluIleAspGlyLysHisPheTyrPheAlaGluAsnGlyGluMet                               260265270                                                                      GlnIleGlyValPheAsnThrGluAspGlyPheLysTyrPheAlaHis                               275280285                                                                      HisAsnGluAspLeuGlyAsnGluGluGlyGluGluIleSerTyrSer                               290295300                                                                      GlyIleLeuAsnPheAsnAsnLysIleTyrTyrPheAspAspSerPhe                               305310315320                                                                   ThrAlaValValGlyTrpLysAspLeuGluAspGlySerLysTyrTyr                               325330335                                                                      PheAspGluAspThrAlaGluAlaTyrIleGlyLeuSerLeuIleAsn                               340345350                                                                      AspGlyGlnTyrTyrPheAsnAspAspGlyIleMetGlnValGlyPhe                               355360365                                                                      ValThrIleAsnAspLysValPheTyrPheSerAspSerGlyIleIle                               370375380                                                                      GluSerGlyValGlnAsnIleAspAspAsnTyrPheTyrIleAspAsp                               385390395400                                                                   AsnGlyIleValGlnIleGlyValPheAspThrSerAspGlyTyrLys                               405410415                                                                      TyrPheAlaProAlaAsnThrValAsnAspAsnIleTyrGlyGlnAla                               420425430                                                                      ValGluTyrSerGlyLeuValArgValGlyGluAspValTyrTyrPhe                               435440445                                                                      GlyGluThrTyrThrIleGluThrGlyTrpIleTyrAspMetGluAsn                               450455460                                                                      GluSerAspLysTyrTyrPheAsnProGluThrLysLysAlaCysLys                               465470475480                                                                   GlyIleAsnLeuIleAspAspIleLysTyrTyrPheAspGluLysGly                               485490495                                                                      IleMetArgThrGlyLeuIleSerPheGluAsnAsnAsnTyrTyrPhe                               500505510                                                                      AsnGluAsnGlyGluMetGlnPheGlyTyrIleAsnIleGluAspLys                               515520525                                                                      MetPheTyrPheGlyGluAspGlyValMetGlnIleGlyValPheAsn                               530535540                                                                      ThrProAspGlyPheLysTyrPheAlaHisGlnAsnThrLeuAspGlu                               545550555560                                                                   AsnPheGluGlyGluSerIleAsnTyrThrGlyTrpLeuAspLeuAsp                               565570575                                                                      GluLysArgTyrTyrPheThrAspGluTyrIleAlaAlaThrGlySer                               580585590                                                                      ValIleIleAspGlyGluGluTyrTyrPheAspProAspThrAlaGln                               595600605                                                                      Leu                                                                            (2) INFORMATION FOR SEQ ID NO:31:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 12 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: Not Relevant                                                 (D) TOPOLOGY: Not Relevant                                                     (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION: 12                                                               (D) OTHER INFORMATION: /note= "The asparagine residue at                       this position contains an amide group."                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                       CysGlnThrIleAspGlyLysLysTyrTyrPheAsn                                           1510                                                                           (2) INFORMATION FOR SEQ ID NO:32:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 5 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: Not Relevant                                                 (D) TOPOLOGY: Not Relevant                                                     (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                       HisHisHisHisHis                                                                15                                                                             __________________________________________________________________________ 

We claim:
 1. A method of treating Clostridium difficile disease, comprising:a) providing:i) a subject exposed to Clostridium difficile exhibiting symptoms comprising diarrhea; and ii) avian antibody reactive with Toxin A of Clostridium difficile, said antibody in a therapeutic amount that is orally administrable, and b) administering said antibody orally to said subject under conditions such that said subject ceases to exhibit symptoms and treatment can be terminated.
 2. The method of claim 1, wherein said subject exhibits long-term survival beyond the treatment period.
 3. The method of claim 1, wherein said avian antibodies reacts with the A-6 interval of Toxin A and wherein said A-6 interval has the sequence set forth in SEQ ID NO:29.
 4. The method of claim 1, further comprising oral administration of an avian antibody reactive with Toxin B of Clostridium difficile.
 5. The method of claim 4, wherein said avian antibodies react with the B-3 interval of Toxin B and wherein said B-3 interval has the sequence set forth in SEQ ID NO:30.
 6. A method of treating Clostridium difficile disease, comprising:a) providing:i) a subject, ii) a first avian antitoxin directed against Clostridium difficile Toxin A, wherein said antitoxin neutralizes Clostridium difficile Toxin A in vivo, and iii) a second avian neutralizing antitoxin directed against Clostridium difficile Toxin B, wherein said antitoxin neutralizes Clostridium difficile Toxin B in vivo; b) mixing said first and second antitoxin to create a therapeutic mixture; and c) administering said therapeutic mixture orally to said subject.
 7. The method of claim 6 further comprising the step of, prior to step c), processing said therapeutic mixture to improve its enteric stability.
 8. The method of claim 7, wherein said processing comprises combining said therapeutic mixture with nutritional formula.
 9. The method of claim 7 wherein said processing comprises encapsulating said antitoxins of said therapeutic mixture.
 10. The method of claim 9 wherein said encapsulating step comprises overcoating with an enteric film.
 11. The method of claim 6 wherein said subject has been exposed to either Clostridium difficile toxin A or toxin B prior to administration of said antitoxin.
 12. The method of claim 11 wherein said subject is suffering from the symptoms of bacterial intoxication and said administering results in the substantial elimination of said symptoms.
 13. The method of claim 12 wherein said symptoms comprise diarrhea.
 14. The method of claim 6 wherein said subject has not been exposed to either Clostridium difficile toxin A or toxin B prior to administration of said antitoxin.
 15. The method of claim 6, wherein said first avian antitoxin is directed against a portion of Clostridium difficile Toxin A sequence SEQ ID NO:6.
 16. The method of claim 15, wherein said portion of Clostridium difficile Toxin A comprises a protein sequence selected from the group comprising SEQ ID NOS:7, 8 and
 29. 17. The method of claim 6, wherein said second avian antitoxin is directed against a portion of Clostridium difficile Toxin B sequence SEQ ID NO:
 10. 18. The method of claim 17, wherein said portion of Clostridium difficile Toxin B comprises a protein sequence selected from the group comprising SEQ ID NOS:20, 21 and
 30. 19. A method of treating Clostridium difficile disease, comprising:a) providing:i) a subject, ii) a first avian antitoxin directed against Clostridium difficile Toxin A, wherein said antitoxin neutralizes Clostridium difficile Toxin A in vivo, and iii) a second avian neutralizing antitoxin directed against Clostridium difficile Toxin B, wherein said antitoxin neutralizes Clostridium difficile Toxin B in vivo; b) mixing said first and second antitoxin to create a therapeutic mixture; c) encapsulating said therapeutic mixture; and d) administering said encapsulated therapeutic mixture orally to said subject.
 20. The method of claim 19 wherein said encapsulating step comprises overcoating with an enteric film.
 21. The method of claim 19 wherein said subject has been exposed to either Clostridium difficile toxin A or toxin B prior to administration of said antitoxin.
 22. The method of claim 21 wherein said subject is suffering from the symptoms of intoxication and said administering results in the substantial elimination of said symptoms.
 23. The method of claim 22 wherein said symptoms comprise diarrhea.
 24. The method of claim 19 wherein said subject has not been exposed to either Clostridium difficile toxin A or toxin B prior to administration of said antitoxin.
 25. The method of claim 19, wherein said first avian antitoxin is directed against a portion of Clostridium difficile Toxin A sequence SEQ ID NO:6.
 26. The method of claim 25, wherein said portion of Clostridium difficile Toxin A comprises a protein sequence selected from the group comprising SEQ ID NOS:7, 8 and
 29. 27. The method of claim 19, wherein said second avian antitoxin is directed against a portion of Clostridium difficile Toxin B sequence SEQ ID NO:
 10. 28. The method of claim 27, wherein said portion of Clostridium difficile Toxin B comprises a protein sequence selected from the group comprising SEQ ID NOS:20, 21 and
 30. 